Identification of genes involved in Alzheimer&#39;s disease using Drosophila melanogaster

ABSTRACT

Transgenic flies displaying altered phenotypes due to expression of the Abeta and C 99  portions of the human APP gene are disclosed. Use of these flies in a method to identify  Drosophila  genes and the human homologs of these  Drosophila  genes, that are potentially involved in Alzheimer&#39;s Disease, is also disclosed. The use of said human homologs as drug targets for the development of therapeutics to treat Alzheimer&#39;s Disease and other conditions associated with defects in the APP pathway, as well as pharmaceutical compositions comprising substances directed to these genes, are also disclosed.

This application claims priority from U.S. Provisional Application60/236,893, filed Sep. 29, 2000, and U.S. Provisional Application60/298,309, filed Jun. 14, 2001, the disclosures of which are hereinincorporated by reference.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a neurological disorder resulting in thedegeneration and eventual death of neurons in brain centers controllingmemory, cognition and behavior. The hallmark of the disease is theformation of insoluble amyloid deposits (senile plaques), the majorcomponent of which is the 40-42 amino acid amyloid beta (Aβ) peptide, aproteolytic product of the amyloid precursor protein (APP). Theseplaques are widely believed to be the major causative agents leading tothe degeneration and death of neuronal cells.

The three major known genes associated with inheritance of familialAlzheimer's disease (FAD) in humans are the transmembrane receptoramyloid precursor protein (APP) and the two preseniln (PS1 and PS2)genes. Missense mutations in these genes result in the increasedproduction of the Aβ (peptide, underscoring the importance of thispeptide in contributing to the disease state. APP is cleaved at twosites, beta and gamma, to release a 40-42 amino acid peptide, Aβ(reviewed in Mills, J. and Reiner, P. B. (1999) J. Neurochem 72:443-460). Missense mutations in APP near the gamma site (Goate, A. etal., (1991). Nature 349: 704-706.), where the C-terminal end of thepeptide is cleaved, result in production of more Aβ 42, by altering the40/42 ratio (Suzuki, N., et al.(994). Science 264:1336-1340). Mutationsaround the beta site result in more overall production of both forms(Mullan, M., et al. (1992). Nat. Genet. 1: 345-347.); Citron, M. et al.(1995). Neuron 14: 661-670).

The presenilins are multiple pass transmembrane proteins, the functionsof which are currently a matter of debate. Missense mutations inpresenilins increase the release of the Aβ 42 form (Borchelt, D. R., etal. (1996). Neuron 17: 1005-1013); Citron, M., et al. (1997). Nat. Med.3: 67-72; Murayama, O. et al. (1999). Neurosci. Lett. 265: 61-63) andaccount for the majority of FAD cases (Sherrington, R., et al.(1995).Nature 375: 754-760).

Many studies have examined the roles of both the soluble and insoluble(aggregated) forms of Aβ and it is widely believed that the aggregatedform of the peptide is responsible for the observed toxic effects (Pike,C. J., et al. (1993). J. Neurosci. 13: 1676-1687; Lorenzo, A. andYankner, B. A. (1994). Proc. Natl. Acad. Sci. USA 91:12243-12247;Giovannelli, L., et al (1998). Neurosci. 87: 349-357). There are anumber of mechanisms that contribute to Aβ-induced death of neurons,including the disruption of intracellular calcium levels (for reviews,see Fraser, S. P., et al. (1997). Trends Neurosci. 20: 67-72; Mattson,M. P. (1997). Physiol. Rev. 77: 1081-1132; Coughlan, C. M. and Breen, K.C. (2000). Pharmacol. and Ther. 86: 111-144), the induction of aninflammatory response caused by activation of microglial cells (reviewedin Coughlan, C. M. and Breen, K. C. (2000). Pharmacol. and Ther. 86:111-144) and the marked degeneration and/or disruption of thebasal-forebrain cholinergic system, which is involved in learning andmemory (reviewed in Hellström-Lindahl and Court, 2000, Behav. Brain Res.113 (1-2): 159-68). Thus, it is clear that the deleterious effects of Aβoverproduction and its contribution to AD are numerous and complex.

Although a great amount of research has been dedicated to the study ofAlzheimer's Disease and its general pathology, the genetic analysis ofhuman neurodegenerative disorders is limited. As a result, the eventsthat trigger the accumulation of beta amyloid, as well as the preciserole of genes such as APP and others suspected to play a part inAlzheimer's Disease, is poorly understood.

Numerous contributions to the establishment of the central role of Aβ inthe manifestation and progression of AD have come from studies in modelsystems. Transgenic mice expressing either wild type. or mutant forms ofAPP exhibit AD pathology, in many cases developing amyloid plaques in anage-dependent fashion and in some cases displaying altered behavior andcognition (for reviews, see Price, D. L., et al (1998). Annu. Rev.Genet. 32: 461-493; van Leuven, F. (2000). Progress in Neurobiol. 61:305-312). Transgenic mice expressing only the Aβ 42 peptide exhibitextensive neuronal degeneration in brain regions normally affected inAD, and 50% die at 12 months of age (LaFerla, F. M. et al. (1995).Nature Genet. 9: 21-30). The neural cells in these mice eventuallyapoptose, followed by astrogliosis and spongiosis. This demonstratesthat Aβ 42 expression is toxic in vivo, and results in neuronaldegeneration and apoptosis.

The use of Drosophila as a model organism has proven to be an importanttool in the elucidation of human neurodegenerative disease pathways(reviewed in Fortini, M and Bonini, N. (2000). Trends Genet. 16:161-167), as the Drosophila genome contains many relevant humanorthologs that are extremely well conserved in function (Rubin, G. M.,et al. (2000). Science 287: 2204-15). For example, Drosophilamelanogaster carries a gene that is homologous to human APP which isinvolved in nervous system function. The gene, APP-like (Appl), isapproximately 40% identical to the neurogenic isoform (695) of the humanAPP gene over three large domains (Rosen et al., PNAS USA 86:2478-2482(1988)) and, like human APP695, is exclusively expressed in the nervoussystem. Flies deficient for the Appl gene show behavioral defects whichcan be rescued by the human APP gene, suggesting that the two genes havesimilar functions in the two organisms (Luo et al., Neuron 9:595-605(1992)).

In addition, Drosophila models of polyglutamine repeat diseases(Jackson, G. R., et al (1998). Neuron 21: 633-642; Kazemi-Esfaijani, P.and Benzer, S. (2000). Science 287: 1837-1840; Femandez-Funez et al.(2000) Nature 408 (6808):101-6, and Parkinson's disease (Feany, M. B.and Bender, W. W. (2000). Nature 404: 394-398) closely mimic the diseasestate in humans, both at the cellular as well as the physiological leveland have been used successfully to identify other genes that play a rolein these diseases. Thus, the power of Drosophila as a model system isdemonstrated in the ability to represent the disease state and toperform large scale genetic screens.

This invention generally relates to a method to identify compounds andgenes acting on the APP pathway in transgenic Drosophila melanogasterectopically expressing genes related to AD. Expression of thesetransgenes can induce visible phenotypes and it is contemplated hereinthat genetic screens disclosed herein may be used to identify genesinvolved in the APP pathway by the identification of mutations thatmodify the induced visible phenotypes. The genes affected by thesemutations will be called herein “genetic modifiers”. It is contemplatedherein that human homologs of genetic modifiers thus identified would beuseful targets for development of therapeutics to treat conditionsassociated with abnormalities in the APP pathway, including, but notlimited to, the development of Alzheimer Disease (AD) therapeutics. Itis also contemplated herein that some of these human homologs might beoccurring on an area of human chromosome 10, shown to be linked toAlzheimer's disease (Bertram et al., Ertekin-Taner et al., Myers et al.,Science 290, 2302-2305, 2000). Such human homologs might have thepotential to be genetically linked to AD and serve as markers for AD oras targets for the development of therapeutics to treat conditionsassociated with abnormalities in the APP pathway, including, but notlimited to, the development of Alzheimer Disease (AD) therapeutics. Suchhuman homologs might also be acting in cellular pathways involving geneslinked to AD and these human homologs might be used to identify thegenes in these pathways.

SUMMARY OF THE INVENTION

The present invention pertains to a transgenic fly whose genomecomprises a DNA sequence encoding a polypeptide comprising the Abetaportion of human APP wherein said DNA sequence encodes Abeta40 (SEQ ID:NO 1) or Abeta42 (SEQ ID:NO 2), fused to a signal sequence, said DNAsequence operably linked to a tissue-specific expression controlsequence; and expressing said DNA sequence, wherein expression of saidDNA sequence results in said fly displaying an altered phenotype. In oneparticular embodiment, the DNA sequence encodes Abeta42, the tissuespecific expression control sequence comprises the eye-specific promoterGMR and expression of the DNA sequence results in an altered phenotypereferred to as the “rough eye” phenotype.

In a further aspect, the invention pertains to a transgenic fly whosegenome comprises a DNA sequence encoding a polypeptide comprising thewild type C99 portion of human APP (SEQ. ID NO:3) or C99 portion ofhuman APP with the London Mutation (SEQ ID NO: 4) fused to a signalsequence, said DNA sequence operably linked to a tissue-specificexpression control sequence; and expressing said DNA sequence, whereinexpression of said DNA sequence results in said fly displaying analtered phenotype. In one embodiment, the DNA sequence encodes the wildtype C99, the tissue-specific expression control sequence is the UAScontrol element, which is activated by Gal4 protein produced in thebrain by the 7B-Gal4 transgene and expression of the DNA sequenceresults in an altered phenotype characterized by a locomotory defect. Inanother particular embodiment, the DNA sequence encodes either the wildtype C99 or the C99 portion of human APP with the London Mutation, thetissue-specific expression control sequence is UAS control elementactivated by Gal4 protein produced by the apterous-Gal4 transgene andexpression of the DNA sequence results in an altered phenotype referredto as the “concave wing” phenotype.

In a further aspect, the invention pertains to a method to identifygenetic modifiers of the APP pathway, said method comprising providing atransgenic fly whose genome comprises a DNA sequence encoding apolypeptide comprising the Abeta portion of human APP wherein said DNAsequence encodes Abeta40 (SEQ ID NO: 1) or Abeta42 (SEQ ID NO: 2), fusedto a signal sequence, said DNA sequence operably linked to atissue-specific expression control sequence; and expressing said DNAsequence, wherein expression of said DNA sequence results in said flydisplaying an altered phenotype; crossing said transgenic fly with a flycontaining a mutation in a known or predicted gene; and screeningprogeny of said crosses for flies that carry said DNA sequence and saidmutation and display modified expression of the transgenic phenotype ascompared to controls. In one embodiment, the DNA sequence encodesAbeta42, the tissue specific expression control sequence comprises theeye-specific promoter GMR and expression of said DNA sequence results insaid fly displaying an altered phenotype referred to as the “rough eye”phenotype.

In a further aspect, the invention pertains to a method to identifygenetic modifiers of the APP pathway, said method comprising: providinga transgenic fly whose genome comprises a DNA sequence encoding apolypeptide comprising the wild type C99 portion of human APP (SEQ. IDNO:3) or C99 portion of human APP with the London Mutation (SEQ ID NO:4) fused to a signal sequence, said DNA sequence operably linked to atissue-specific expression control sequence; and expressing said DNAsequence, wherein expression of said DNA sequence results in said flydisplaying an altered phenotype; crossing said transgenic fly with a flycontaining a mutation in a known or predicted gene; and, screeningprogeny of said crosses for flies that carry said DNA sequence and saidmutation and display modified expression of the transgenic phenotype ascompared to controls. In one embodiment, the DNA sequence encodes thewild type C99, the tissue-specific expression control sequence is theUAS control element, activated by Gal4 protein produced in the brain bythe 7B-Gal4 transgene and expression of said DNA sequence results insaid fly displaying an altered phenotype characterized by a locomotorydefect. In another embodiment, the DNA sequence encodes either the wildtype C99 or the C99 portion of human APP with the London Mutation, thetissue-specific expression control sequence is UAS control elementactivated by Gal4 protein produced by the apterous-Gal4 transgene andexpression of said DNA sequence results in said fly displaying analtered phenotype referred to as the. “concave wing” phenotype.

A further aspect of the invention pertains to a method to identifycompounds that act on gene products involved in the APP pathway byassaying for compounds that can modify the phenotypes induced byexpression of Abeta, said method comprising: providing a transgenic flywhose genome comprises a DNA sequence encoding a polypeptide comprisingthe Abeta portion of human APP wherein said DNA sequence encodes Abeta40(SEQ ID NO: 1) or Abeta42 (SEQ ID NO: 2), fused to a signal sequence,said DNA sequence operably linked to a tissue-specific expressioncontrol sequence; and expressing said DNA sequence, wherein expressionof said DNA sequence results in said fly displaying an alteredphenotype; administering to said fly a candidate compound; and, assayingfor changes in the phenotype of said fly as compared to the phenotype ofa similar transgenic fly not administered the candidate compound. In oneembodiment, the DNA sequence encodes Abeta42, the tissue specificexpression control sequence is the eye-specific promoter GMR andexpression of said DNA sequence results in said fly displaying analtered phenotype referred to as the “rough eye” phenotype.

Yet another aspect of the invention pertains to a method to identifycompounds that act on gene products involved in the APP pathway byassaying for compounds that can modify the phenotypes induced byexpression of C99, said method comprising: providing a transgenic flywhose genome comprises a DNA sequence encoding a polypeptide comprisingthe wild type C99 portion of human APP (SEQ. ID NO:3) or C99 portion ofhuman APP with the London Mutation (SEQ ID NO: 4) fused to a signalsequence, said DNA sequence operably linked to a tissue-specificexpression control sequence; and expressing said DNA sequence, whereinexpression of said DNA sequence results in said fly displaying analtered phenotype; administering to said fly a candidate compound; and,assaying for changes in the phenotype of said fly as compared to thephenotype of a similar transgenic fly not administered the candidatecompound. In one embodiment, the DNA sequence encodes wild type C99, thetissue-specific expression control sequence is the UAS control elementactivated by Gal4 protein produced in the brain by the 7B-Gal4 transgeneand expression of said DNA sequence results in said fly displaying aphenotype characterized as a locomotory defect. In another embodiment,the DNA sequence encodes either wild type C99 or the C99 portion ofhuman APP with the London Mutation, the tissue-specific expressioncontrol sequence is UAS control element activated by Gal4 proteinproduced by the apterous-Gal4 transgene and expression of said DNAsequence results in said fly displaying an altered phenotype referred toas the “concave wing” phenotype.

In yet another aspect, the invention pertains to a method foridentifying genes involved in the onset or progression of conditionsassociated with abnormal regulation of the APP pathway, including butnot limited to Alzheimer's Disease, and whose protein products mightserve as potential markers for Alzheimer's Disease, said methodcomprising identifying the human homologs of fly genes that have beenidentified as genetic modifiers according to the methods of the presentinvention.

In yet another aspect, the invention pertains to a method foridentifying genes involved in the onset or progression of conditionsassociated with abnormal regulation of the APP pathway, including butnot limited to Alzheimer's Disease, and whose protein products mightserve as potential markers for Alzheimer's Disease, said methodcomprising identifying human homologs of fly genetic modifier genes thatare located close to the area of human chromosome 10 that is shown tohave genetic linkage to Alzheimer's Disease.

In yet another aspect, the invention pertains to a method foridentifying genes involved in the onset or progression of Alzheimer'sDisease and whose protein products might serve as potential markers forAD, said method comprising identifying genes that are involved in thepathways regulated by the transcription factors encoded by the humansequences hCP50765 (SEQ ID NO. 35, encoded by the EGR2 gene), andhCP41313 (Seq ID NO 15, SEQ ID NO17 or SEQ ID NO 53, encoded by thehuman homolog of the Drosophila nocA gene), which human sequences arelocated close to the area of human chromosome 10 that is shown to havegenetic linkage to Alzheimer's Disease.

In yet another aspect, the invention pertains to a method foridentifying compounds useful for the treatment, prevention oramelioration of pathological conditions associated with defects in theAPP pathway, including but not limited Alzheimer's Disease, comprisingadministering candidate compounds to an in vitro or in vivo model ofAlzheimer's Disease; and assaying for changes in expression of a genetichomolog of a genetic modifier, wherein altered expression of any one ofsaid homologs compared to levels in a control to which a candidatecompound has not been administered indicates a compound of potentialtherapeutic value.

The invention also pertains to a method for the treatment, prevention oramelioration of pathological conditions associated with defects in theAPP pathway, including, but not limited to Alzheimer's Disease,comprising administering to a subject in need thereof a therapeuticallyeffective amount of a compound that may inhibit or promote the functionof any one or more of the polypeptide encoded by the human homologs ofthe genetic modifiers identified herein.

The invention also pertains to a method for the treatment, prevention oramelioration of pathological conditions associated with defects in theregulation of the APP pathway, including but not limited to Alzheimer'sDisease, comprising administering to a subject in need thereof atherapeutically effective amount of a pharmaceutical compositioncomprising any one or more substances selected from the group consistingof: triple helix DNA, antisense oligonucleotides or ribozymes, allcomplementary to the appropriate sequence of a mRNA deriving from anyone or more of the human homologs of genetic modifier genes identifiedaccording to the methods of the present invention.

The invention also pertains to a method for the treatment, prevention oramelioration of pathological conditions associated with defects in theregulation of the APP pathway, including but not limited to Alzheimer'sDisease, comprising administering to a subject in need thereof atherapeutically effective amount of a pharmaceutical compositioncomprising double stranded RNA molecules directed to one or more of thehuman homologs of the genetic modifiers identified according to themethods of the present invention.

In a further aspect, the invention pertains to a method for thetreatment, prevention or amelioration of pathological conditionsassociated with defects in the APP pathway, including but not limited toAlzheimer's Disease, comprising administering to a subject in needthereof a therapeutically effective amount of a pharmaceuticalcomposition comprising an antibody or antibodies and/or fragmentsthereof directed to the polypeptide encoded by any one or more of thehuman homolog of the genetic modifiers identified according to themethods of the present invention.

In a further aspect, the invention also pertains to a method for thediagnosis of pathological conditions associated with abnormalities inthe APP pathway in a subject, including but not limited to Alzheimer'sDisease, which comprises measuring the mRNA level or the level oractivity of the polypeptides encoded by any one or more of the humanhomologs of a genetic modifier in a biological sample from a subject,wherein an abnormal level relative to the level thereof in a controlsubject is diagnostic of said conditions.

In a still further aspect, the invention pertains to a kit comprisingthe components necessary to detect expression levels of polypeptidesencoded by any one or more of the human homologs of a genetic modifieror fragments thereof or polynucleotides encoding any one or more of saidpolypeptides or fragments thereof, in a biological sample from asubject, such kits comprising antibodies that bind to said polypeptidesor to said fragments thereof, or oligonucleotide probes that hybridizewith said polynucleotides or to said fragments thereof and instructionsfor using said kit.

In yet another aspect, the invention pertains to a pharmaceuticalcomposition comprising substances selected from the group consisting of:antisense, ribozyme, double stranded RNA or triple helix nucleic acidsdirected to any one or more of the human homologs of a genetic modifieror fragments thereof, polypeptides encoded by any one or more of thehuman homologs of a genetic modifiers or fragments thereof,polynucleotides encoding said polypeptides or fragments thereof, andantibodies that bind to said polypeptides or fragments thereof, inconjunction with a suitable pharmaceutical carrier, excipient ordiluent, for the treatment of pathological conditions associated withabnormalities in the APP pathway, including but not limited to,Alzheimer's Disease.

The invention also pertains to a method for the treatment ofpathological conditions associated with abnormalities in APP pathwayincluding but not limited to, Alzheimer's Disease, comprisingintroducing nucleic acids encoding any one or more of the human homologsof a genetic modifier into one or more tissues of a subject in needthereof resulting in that one or more proteins encoded by the nucleicacids are expressed and or secreted by cells within the tissue.

DETAILED DESCRIPTION OF THE INVENTION

All patent applications, patents, literature and website referencescited herein are hereby incorporated by reference in their entirety.

In practicing the present invention, many conventional techniques inmolecular biology and recombinant DNA are used. These techniques arewell known and are explained in, for example, Current Protocols inMolecular Biology, Volumes I, II, and III, 1997 (F. M. Ausubel ed.);Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;DNA Cloning: A Practical Approach, Volumes I and II, 1985 (D. N. Glovered.); A Practical Guide to Molecular Cloning; the series, Methods inEnzymology (Academic Press, Inc.); Gene Transfer Vectors for MammalianCells, 1987 (J. H. Miller and M. P. Calos eds., Cold Spring HarborLaboratory); and Methods in Enzymology Vol. 154 and Vol. 155 (Wu andGrossman, and Wu, eds., respectively). Well known Drosophila moleculargenetics techniques can be found, for example, in Robert, D. B.,Drosophila, A Practical Approach (IRL Press, Washington, D.C., 1986).Descriptions of flystocks can be found in the Flybase data base athttp://flybase.bio.indiana.edu.

A “transgenic” organism as used herein refers to an organism that hashad extra genetic material inserted into its genome. As used herein, a“transgenic fly” includes embryonic, larval and adult forms ofDrosophila melanogaster that contain a DNA sequence from the same oranother organism randomly inserted into their genome. AlthoughDrosophila melanogaster is preferred, it is contemplated that any fly ofthe genus Drosophila may be used in the present invention.

As used herein, “ectopic” expression of the transgene refers toexpression of the transgene in a tissue or cell or at a specificdevelopmental stage where it is not normally expressed.

As used herein, “phenotype” refers to the observable physical orbiochemical characteristics of an organism as determined by both geneticmakeup and environmental influences.

As used herein, a compound that may “inhibit or promote the function ofany or more of the polypeptides encoded by the human homolog of agenetic modifier” includes compounds that may do so indirectly (via downstream effects) or directly, by binding to or otherwise interacting withthe protein and includes, but is not limited to, antagonists or agonistsof the protein.

As used herein, a “stringent ortholog” is defined as meeting thefollowing criteria: fly protein X has best match with human protein Yand fly protein X does not have a better match with another fly proteinthan with human protein Y and human protein Y has best match with flyprotein X and human protein Y has no better match with another humanprotein than with fly protein X, wherein “X” and “Y” stand for any twofly and human proteins being compared.

A “putative ortholog” is defined herein as meeting only the followingtwo criteria: fly protein X has best match with human protein Y andhuman protein Y has best match with fly protein X, regardless of whetherfly protein X had a better match with another fly protein and/or whetherhuman protein Y had a better match with another human protein. Asdisclosed herein, all other human/fly protein matches are deemed“homologs”.

As used herein, the term “expression control sequence” refers topromoters and enhancers. The term “promoter” refers to DNA sequenceswhich are recognized directly or indirectly and bound by a DNA-dependentRNA polymerase during the initiation of transcription and includesenhancer elements. Enhancers used in the present invention include theUAS element which is activated by the yeast Gal4 transcriptionalregulator.

The term “transcription factor” refers to any protein required toinitiate or regulate transcription in eukaryotes. For example, theeye-specific promoter GMR is a binding site for the eye-specifictranscription factor, GLASS (Moses, K and Rubin, G M Genes Dev.5(4):583-93 (1991)).

As used herein, the term “Abeta” (Aβ) refers to beta amyloid peptidewhich is a short (40-42 amino acid) peptide produced by proteolyticcleavage of APP by beta and gamma secretases. It is the primarycomponent of amyloid depositions, the hallmark of AD and the cause ofneuronal cell death and degeneration. Abeta peptide of the presentinvention includes, but is not limited to, peptides of 40 and 42 aminoacids and are referred to, respectively, as Abeta40 (or Aβ40) (SEQ IDNO: 1) and Abeta42 (or Aβ42) (SEQ ID NO: 2).

“C99” refers to a peptide that contains the Abeta region plus thecytoplasmic tail of APP (SEQ ID NO: 3). As used herein, the term alsoincludes the C99 London sequence, which carries the London FADAlzheimer's associated mutation (SEQ ID NO:4) (Goate, A., et al (1991).Nature 349: 704-706). Abeta and C99 peptides are well known to one ofskill in the art (see, for example, Golde et al., Science 255:728-730(1992); Coughlan, C. M. and Breen, K. C. (2000). Pharmacol. and Ther.86: 111-144).

“UAS” region as used herein refers to an upstream activating sequencerecognized by the GAL-4 transcriptional activator.

As used herein, a “signal sequence” refers to a short sequence of aminoacids that determines the eventual location of a protein in a cell, forexample, the N-terminal sequence or 20 or so amino acids that directsnascent secretory and transmembrane proteins to the endoplasmicreticulum. It is contemplated herein that any conventional signalsequence familiar to one of skill in the art may be used to ensuretransfer of the encoded C99 or Abeta proteins through the secretorypathway, including, but not limited to, the signal sequence ofendogenous Drosophila Appl or presenilin, or of the windbeutel gene,encoding for a ER (endoplasmic reticulum) resident protein (Konsolakiand Schupbach, Genes & Dev. 12: 120-131 (1998)), or the humanpre-proenkephaline gene signal (SEQ ID NO: 5).

As used herein, a “control” fly refers to a larva or fly that is of thesame genotype as larvae or flies used in the methods of the presentinvention except that the control larva or fly does not carry themutation being tested for modification of phenotype, or is notadministered candidate compounds.

As used herein, a “control subject” refers to an organism that does notsuffer from a condition associated with abnormalities in the APPpathway.

As used herein, a “Drosophila transformation vector” is a DNA plasmidthat contains transposable element sequences and can mediate integrationof a piece of DNA in the genome of the organism. This technology isfamiliar to one of skill in the art.

As the term is used herein, the “rough eye” phenotype is characterizedby disorganization of ommatidia and inter-ommatidial bristles and can becaused by degeneration of neuronal cells. This phenotype is visiblethrough a dissecting stereo-microscope.

As the term is used herein, the “concave wing” phenotype ischaracterized by abnormal folding of the fly wing such that the wingsare bent upwards along their long margins.

As used herein, a “locomotory defect” refers to a phenotype whereinflies display impaired responses to mechanical agitation compared towild type flies in conventional locomotory activity assays.

As used herein, the following and related phrases, pathologicalconditions associated with abnormalities in the APP pathway, conditionsassociated with abnormal regulation of the APP pathway, conditionsrelated to Alzheimer's Disease, pathological conditions associated withdefects in the APP pathway, all include, but are not limited to,Alzheimer's Disease, and include those conditions characterized bydegeneration and eventual death of neurons in brain clusters controllingmemory, cognition and behavior.

“Therapeutically effective amount” refers to that amount of activeingredient, for example compound or gene product which ameliorates thesymptoms of the condition being treated.

Methods of obtaining transgenic organisms, including transgenicDrosophila, are well known to one skilled in the art. For example, acommonly used reference for P-element mediated transformation isSpradling, 1986. P element mediated transformation. In Drosophila: Apractical approach (ed. D. B. Roberts), pp 175-197. IRL Press, Oxford,UK)). The EP element technology refers to a binary system, utilizing theyeast Gal4 transcriptional activator, that is used to ectopicallyregulate the transcription of endogenous Drosophila genes. Thistechnology is described in : Brand and Perrimon, 1993. Targeted geneexpression as a means of altering cell fates and generating dominantphenotypes. Development 118, pp 410-415 and in: Rorth et al, 1998.Systematic gain-of-function genetics in Drosophila. Development, 125(6),pp 1049-1057.

The present invention discloses a transgenic fly, Drosophilamelanogaster, that contains in its genome a DNA sequence encoding apolypeptide comprising the beta amyloid portion (SEQ ID NO: 1 or SEQ IDNO: 2) or C99 portion of the human APP gene (SEQ ID NO: 3 or SEQ ID NO:4) which is fused at its N-terminus according to conventional methods toa signal peptide sequence, for example, SEQ ID NO:5, to ensure transferof the encoded polypeptide through the secretory pathway. The fused DNAsequences are operably linked to tissue-specific expression controlsequences such as promoter regions or upstream activating sequences(UAS), depending on the expression system utilized. These expressioncontrol sequences include those that are specific for neural tissue inthe fly and include organs such as the eye, wing, notum, brain, CNS andPNS. Under the control of these tissue specific control sequences,encoded peptides are transcribed to form mRNA which is translated intodetectable levels of beta amyloid or C99 peptide and which causesaltered phenotypes in the flies. By assaying for changes in thesephenotypes, these flies can be used to identify genes or compounds thatmay affect the APP pathway and may provide insight into the molecularand biochemical mechanisms of the APP pathway and Alzheimer's Disease.

Conventional expression control systems may be used to achieve ectopicexpression of proteins of interest, including the beta amyloid and C99peptides of the present invention. Such expression may result in thedisturbance of biochemical pathways and the generation of alteredphenotypes. One such expression control system involves direct fusion ofthe DNA sequence to expression control sequences of tissue-specificallyexpressed genes, such as promoters or enhancers. Another expressioncontrol system that may be used is the binary Gal4-transcriptionalactivation system (Brand and Perrimon, Development 118:401-415 (1993)).

The Gal4 system uses the yeast transcriptional activator Gal4, to drivethe expression of a gene of interest in a tissue specific manner. TheGal4 gene has been randomly inserted into the fly genome, using aconventional transformation system, so that it has come under thecontrol of genomic enhancers that drive expression in a temporal andtissue-specific manner. Individual strains of flies have beenestablished, called “drivers”, that carry those insertions (Brand andPerrimon, Development 118:401-415 (1993)).

In the Gal4 system, a gene of interest is cloned into a transformationvector, so that its transcription is under the control of the UASsequence (upstream Activating Sequence), the Gal4-responsive element.When a fly strain that carries the UAS-gene of interest sequence iscrossed to a fly strain that expresses the Gal4 gene under the controlof a tissue specific enhancer, the gene will be expressed in a tissuespecific pattern.

In order to generate phenotypes that are easily visible in adult tissuesand can thus be used in genetic screens, Gal4 “drivers” that driveexpression in later stages of the fly development may be used in thepresent invention. Using these drivers, expression would result inpossible defects in the wings, the eyes, the legs, different sensoryorgans and the brain. These “drivers” include, for example,apterous-Gal4 (wings), elav-Gal4 (CNS), sevenless-Gal4, eyeless-Gal4 andpGMR-Gal4 (eyes). In addition, since Appl, the fly homologue of APP, isexclusively expressed in neural tissue, “driver” strains in which atleast a subset of expression is directed to a part of the nervoussystem, are preferred. This includes the brain specific 7B-Gal4 driver.Descriptions of the Gal4 lines and notes about their specific expressionpatterns is available in Flybase (http://flybase.bio.indiana.edu) .

Various DNA constructs may be used to generate the transgenic Drosophilamelanogaster of the present invention. For example, the construct maycontain the beta amyloid or C99 portion of the human APP gene fused tothe pre-proenkephaline gene signal peptide sequence and operably linkedto the eye-specific promoter, GMR. In another example, the construct maycontain the beta amyloid portion or C99 of the human APP gene fused tothe human pre-proenkephaline gene signal peptide sequence cloned intothe pUAST vector (Brand and Perrimon, Development 118:401-415 (1993))which places the UAS sequence upstream of the transcribed region.Insertion of these constructs into the fly genome may occur throughP-element recombination, Hobo element recombination (Blackman et al.,EMBO J. 8:211-217 (1989)), homologous recombination (Rong and Golic,Science 288:2013-2018 (2000)) or other standard techniques known to oneof skill in the art.

As discussed above, an ectopically expressed gene may result in analtered phenotype by disruption of a particular biochemical pathway.Mutations in genes acting in the same biochemical pathway are expectedto cause modification of the altered phenotype. Thus, the flies of thepresent invention can be used to identify genes acting in the APPpathway by crossing a C99 or Abeta transgenic fly with a fly containinga mutation in a known or predicted gene; and screening progeny of thecrosses for flies that display quantitative or qualitative modificationof the altered phenotype of the C99 or Abeta transgenic fly, as comparedto controls. Thus, this system is extremely beneficial for theelucidation of the function of processed APP gene products, as well asthe identification of other genes that directly or indirectly interactwith them. Mutations that can be screened include, but are not limitedto, loss-of-function alleles of known genes, deletion strains,“enhancer-trap” strains generated by the P-element and gain-of-functionmutations generated by random insertions into the Drosophila genome of aGal4-inducible construct that can activate the ectopic expression ofgenes in the vicinity of its insertion. It is contemplated herein thatgenes involved in the APP pathway can be identified in this manner andthese genes can then serve as targets for the development oftherapeutics to treat conditions associated with abnormalities in theAPP pathway, leading to diseases, including but not limited to,Alzheimer's Disease.

The C99 and Abeta transgenic flies of the present invention may also beused in a method to identify compounds that may modify the APP pathwayand may thus prove useful for the treatment of conditions discussedabove. Said method may comprise administering candidate compounds to C99or Abeta transgenic flies and then assaying for changes in the phenotypeof the C99 or Abeta transgenic fly as compared to the phenotype ofcontrol C99 or Abeta transgenic flies that have not been administeredthe compound. For example, using conventional methods, candidatecompounds can be fed to larvae expressing a beta amyloid or C99. Thelarvae can then be grown to the adult stage and modification of the C99or Abeta-induced phenotype assayed. Candidate compounds may also be fedto adult flies and modifications of phenotype assayed.

The mechanism of action of compounds thus identified may be examined bycomparing the phenotypes produced by genetic manipulation with thoseinduced by the administration of a compound of interest. Such compoundsinclude those that may ameliorate or worsen the altered phenotypecreated in the transgenic flies. Expression of a compound-inducedphenotype similar to one associated with a known genetic modificationwould suggest that the compound has an effect on the same pathway thatthe genetic modification is affecting.

In addition to screening compounds in the transgenic flies of thepresent invention, such compounds may also be further assayed byemploying in vitro and other in vivo models of AD using conventionalmethods. For example, numerous cell lines may be used as in vitro modelsof AD and are familiar to one of skill in the art, including, forexample, the cell lines described in Xia et al, 1997 PNAS USA 94(15):8208-13. In vivo models also exist and include, for example, themouse model of AD disclosed in WO 94/00569.

Elucidation of the mechanism of action of compounds which affect theaction of beta amyloid or C99 in the transgenic flies disclosed hereinmay also be performed using RNA profiling on chips (Affymetrix, SantaClara) or using other conventional methods. For example, the RNAprofiles of flies which have been administered candidate compounds maybe assayed and compared to those of flies which have been geneticallymodified. Similar profiles would suggest that the compound acts in someway on the beta amyloid or C99 affected pathway.

It is contemplated herein that, in yet another aspect, the inventionpertains to a method for identifying genes involved in the onset orprogression of Alzheimer's Disease and whose protein products mightserve as potential markers for AD, said method comprising identifyinggenes that are involved in the pathways regulated by the transcriptionfactors encoded by the human sequences hCP50765 (SEQ ID NO. 35, encodedby the EGR2 gene), and hCP41313 (Seq ID NO 15, SEQ ID NO17 or SEQ ID NO53, encoded by the human homolog of the Drosophila nocA gene), whichhuman sequences are homologs of Drosophila genetic modifiers identifiedas described herein and are located close to the area of humanchromosome 10 that is shown to have genetic linkage to Alzheimer'sDisease. Identification of such genes, regulated by the above mentionedtranscription factors, may be achieved using conventional methods,including but not limited to, a technology called SELEX, referenced inTuerk and Gold, 1990, Science 249, 505-510 and Brown and Gold, 1995,Biochemistry 34, 14765-14774. For example, genes that are regulated by aspecific transcription factor can be identified by determining thetarget DNA sequence of the specific transcription factor. Such targetsequence identification can be achieved by different methods, includingbut not limited to SELEX. Once the target sequence is identified, thepresence of this sequence in the upstream regulatory regions of knownand predicted genes can be determined, using bio-informatics tools wellknown to one of skill in the art. Genes containing the target sequencein their upstream regulatory regions can be expected to be regulated bythe specific transcription factor.

It is contemplated that compounds which can affect (e.g. inhibit orpromote) the function or expression of proteins encoded by the humanhomologs of genetic modifiers identified according to the presentinvention may be useful to treat Alzheimer's Disease or other conditionsassociated with defects in the regulation of the APP pathway. Inaddition, it is also contemplated that, using conventional methods,antisense oligonucleotides, ribozymes, triple helix DNA and/or doublestranded RNA of therapeutic value may be created based on the nucleotidesequences of these human homologs of genetic modifiers. The therapeuticuse of antibodies directed to the polypeptides encoded by human homologsof genetic modifiers and created using conventional methods is alsocontemplated herein. Thus, an additional aspect of the invention relatesto the administration of a pharmaceutical composition, in conjunctionwith a pharmaceutically acceptable carrier, excipient or diluent, forthe treatment of Alzheimer's Disease and related conditions. Suchpharmaceutical compositions may comprise the compounds, antisenseoligonucleotides, ribozymes, triple helix DNA, double stranded RNAand/or antibodies discussed above. The compositions may also containexpression products of human homologs of the genetic modifiers (e.g.polypeptides or fragments thereof) identified according to the presentinvention. The compositions may be administered alone or in combinationwith at least one other agent, such as stabilizing compound, which maybe administered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a subject in need thereofalone, or in combination with other agents, drugs or hormones.

The pharmaceutical compositions encompassed by the invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-articular, intra-arterial,intramedullary, intrathecal, intraventricular, transdermal,subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual,or rectal means.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated m aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Non-lipid polycationicamino polymers may also be used for delivery. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0. 1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of the compounds or gene. productsidentified according to the present invention, such labeling wouldinclude amount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A “therapeutically effective dose” refers to that amount of activeingredient, for example compound or gene product which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between toxic and therapeutic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc. Pharmaceutical formulations suitable fororal administration of proteins are described, e.g., in U.S. Pat. Nos.5,008,114; 5,505,962; 5,641,515; 5,681,811; 5,700,486; 5,766,633;5,792,451; 5,853,748; 5,972,387; 5,976,569; and 6,051,561.

It is also contemplated herein that a method for the diagnosis ofpathological conditions associated with abnormalities in the APP pathwayin a subject, including but not limited to Alzheimer's Disease, ispossible given the data of Table 1 For example, the method may comprisemeasuring the level of polypeptides encoded by any one or more of thehuman genetic homologs of the genes of Table 1 in a biological samplefrom a subject, wherein an abnormal level of any one or more of saidpolypeptides relative to the level thereof in a normal subject isdiagnostic of said conditions. Such an assay could be performed usingconventional technologies familiar to one of skill in the art.

In another embodiment, nucleic acids comprising a sequence encoding ahuman homolog of a genetic modifier or functional derivative thereof areadministered to promote APP pathway function, by way of gene therapy.Gene therapy refers to therapy performed by the administration of anucleic acid to a subject. In this embodiment of the invention, thenucleic acid produces its encoded protein that mediates a therapeuticeffect by promoting normal APP pathway function.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

In a preferred aspect, the therapeutic comprises the nucleic acid for agenetic modifier that is part of an expression vector that expresses agenetic modifier protein or fragment or chimeric protein thereof in asuitable host. In particular, such a nucleic acid has a promoteroperably linked to the specific genetic modifier protein coding region,said promoter being inducible or constitutive, and, optionally,tissue-specific. In another particular embodiment, a nucleic acidmolecule is used in which the modifier protein coding sequences and anyother desired sequences are flanked by regions that promote homologousrecombination at a desired site in the genome, thus providing forintrachromosomal expression of the modifier nucleic acid (Koller andSmithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra etal., 1989, Nature 342:435-438).

Delivery of the nucleic acid into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vector, or indirect, in which case, cells arefirst transformed with the nucleic acid in vitro, then transplanted intothe patient. These two approaches are known, respectively, as in vivo orex vivo gene therapy.

In a specific embodiment, the nucleic acid is directly administered invivo, where it is expressed to produce the encoded product. This can beaccomplished by any of numerous methods known in the art, e.g., byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular, e.g., byinfection using a defective or attenuated retroviral or other viralvector (see, e.g., U.S. Pat. No. 4,980,286 and others mentioned infra),or by direct injection of naked DNA, or by use of microparticlebombardment (e.g., a gene gun; Biolistic, Dupont), or coating withlipids or cell-surface receptors or transfecting agents, encapsulationin liposomes, microparticles, or microcapsules, or by administering itin linkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see e.g., U.S. Pat. Nos. 5,166,320; 5,728,399; 5,874,297;and 6,030,954, all of which are incorporated by reference herein intheir entirety) (which can be used to target cell types specificallyexpressing the receptors), etc. In another embodiment, a nucleicacid-ligand complex can be formed in which the ligand comprises afusogenic viral peptide to disrupt endosomes, allowing the nucleic acidto avoid lysosomal degradation. In yet another embodiment, the nucleicacid can be targeted in vivo for cell specific uptake and expression, bytargeting a specific receptor (see, e.g., PCT Publications WO 92/06180;WO 92/22635; WO92/20316; WO93/14188; and WO 93/20221). Alternatively,the nucleic acid can be introduced intracellularly and incorporatedwithin host cell DNA for expression, by homologous recombination (see,e.g., U.S. Pat. Nos. 5,413,923; 5,416,260; and 5,574,205; and Zijlstraet al., 1989, Nature 342:435-438).

In a specific embodiment, a viral vector that contains a modifiernucleic acid is used. For example, a retroviral vector can be used (see,e.g., U.S. Pat. Nos. 5,219,740; 5,604,090; and 5,834,182). Theseretroviral vectors have been modified to delete retroviral sequencesthat are not necessary for packaging of the viral genome and integrationinto host cell DNA. The modifier nucleic acid to be used in gene therapyis cloned into the vector, which facilitates delivery of the gene into apatient.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Methods for conductingadenovirus-based gene therapy are described in, e.g., U.S. Pat. Nos.5,824,544; 5,868,040; 5,871,722; 5,880,102; 5,882,877; 5,885,808;5,932,210; 5,981,225; 5,994,106; 5,994,132; 5,994,134; 6,001,557; and6,033,8843, all of which are incorporated by reference herein in theirentirety.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy. Methods for producing and utilizing AAV are described, e.g., inU.S. Pat. Nos. 5,173,414; 5,252,479; 5,552,311; 5,658,785; 5,763,416;5,773,289; 5,843,742; 5,869,040; 5,942,496; and 5,948,675, all of whichare incorporated by reference herein in their entirety.

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells and may be used in accordance with the presentinvention, provided that the necessary developmental and physiologicalfunctions of the recipient cells are not disrupted. The technique shouldprovide for the stable transfer of the nucleic acid to the cell, so thatthe nucleic acid is expressible by the cell and preferably heritable andexpressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. In a preferred embodiment, epithelial cellsare injected, e.g., subcutaneously. In another embodiment, recombinantskin cells may be applied as a skin graft onto the patient. Recombinantblood cells (e.g., hematopoietic stem or progenitor cells) arepreferably administered intravenously. The amount of cells envisionedfor use depends on the desired effect, patient state, etc., and can bedetermined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy, amodifier nucleic acid is introduced into the cells such that it isexpressible by the cells or their progeny, and the recombinant cells arethen administered in vivo for therapeutic effect. In a specificembodiment, stem or progenitor cells are used. Any stem-and/orprogenitor cells that can be isolated and maintained in vitro canpotentially be used in accordance with this embodiment of the presentinvention. Such stem cells include but are not limited to hematopoieticstem cells (HSC), stem cells of epithelial tissues such as the skin andthe lining of the gut, embryonic heart muscle cells, liver stem cells(see, e.g., WO 94/08598), and neural stem cells (Stemple and Anderson,1992, Cell 71:973-985).

Epithelial stem cells (ESCs) or keratinocytes can be obtained fromtissues such as the skin and the lining of the gut by known procedures(Rheinwald, 1980, Meth. Cell Bio. 21A:229). In stratified epithelialtissue such as the skin, renewal occurs by mitosis of stem cells withinthe germinal layer, the layer closest to the basal lamina. Stem cellswithin the lining of the gut provide for a rapid renewal rate of thistissue. ESCs or keratinocytes obtained from the skin or lining of thegut of a patient or donor can be grown in tissue culture (Pittelkow andScott, 1986, Mayo Clinic Proc. 61:771). If the ESCs are provided by adonor, a method for suppression of host versus graft reactivity (e.g.,irradiation, drug or antibody administration to promote moderateimmunosuppression) can also be used.

With respect to hematopoietic stem cells (HSC), any technique thatprovides for the isolation, propagation, and maintenance in vitro of HSCcan be used in this embodiment of the invention. Techniques by whichthis may be accomplished include (a) the isolation and establishment ofHSC cultures from bone marrow cells isolated from the future host, or adonor, or (b) the use of previously established long-term HSC cultures,which may be allogeneic or xenogeneic. Non-autologous HSC are usedpreferably in conjunction with a method of suppressing transplantationimmune reactions of the future host/patient. In a particular embodimentof the present invention, human bone marrow cells can be obtained fromthe posterior iliac crest by needle aspiration (see, e.g., Kodo et al.,1984, J. Clin. Invest. 73:1377-1384). In a preferred embodiment of thepresent invention, the HSCs can be made highly enriched or insubstantially pure form. This enrichment can be accomplished before,during, or after long-term culturing, and can be done by any techniquesknown in the art. Long-term cultures of bone marrow cells can beestablished and maintained by using, for example, modified Dexter cellculture techniques (Dexter et al., 1977, J. Cell Physiol. 91:335) orWitlock-Witte culture techniques (Witlock and Witte, 1982, Proc. Natl.Acad. Sci. USA 79:3608-3612).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

A further embodiment of the present invention relates to the therapeuticuse of a purified antibody or a fragment thereof for the treatment ofconditions associated with abnormalities in the APP pathway, includingbut not limited to, AD. It is contemplated that the purified antibody ora fragment thereof specifically binds to a polypeptide that comprisesthe amino acid sequence of any of the human homologs of the geneticmodifiers identified in Table 1, preferably, the polypeptides of humanhomologs located on chromosome 10 disclosed herein, most preferably, thepolypeptide encoded by SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO 53,i.e. the curated noc A sequences, or to a fragment of said polypeptides.A preferred embodiment relates to a fragment of such an antibody, whichfragment is an Fab or F(ab′)₂ fragment. In particular, the antibody canbe a polyclonal antibody or a monoclonal antibody.

Described herein are methods for the production of antibodies capable ofspecifically recognizing one or more differentially expressed geneepitopes. Such antibodies may include, but are not limited to polyclonalantibodies, monoclonal antibodies (mAbs), humanized or chimericantibodies, single chain antibodies, Fab fragments, F(ab′)₂ fragments,fragments produced by a Fab expression library, anti-idiotypic (anti-Id)antibodies, and epitope-binding fragments of any of the above. Suchantibodies may be used, for example, in the detection of a fingerprint,target, gene in a biological sample, or, alternatively, as a method forthe inhibition of abnormal target gene activity. Thus, such antibodiesmay be utilized as part of Alzheimer's disease treatment methods, and/ormay be used as part of diagnostic techniques whereby patients may betested for abnormal levels of a modifier polypeptide, or for thepresence of abnormal forms of a modifier polypeptide.

For the production of antibodies to a specific modifier polypeptide,various host animals may be immunized by injection with the polypeptide,or a portion thereof.. Such host animals may include but are not limitedto rabbits, mice, and rats, to name but a few. Various adjuvants may beused to increase the immunological response, depending on the hostspecies, including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentiallyuseful human adjuvants such as BCG (bacille Calmette-Guerin) andCorynebacterium parvum.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as target gene product, or an antigenic functional derivativethereof. For the production of polyclonal antibodies, host animals suchas those described above, may be immunized by injection with a modifierpolypeptide, or a portion thereof, supplemented with adjuvants as alsodescribed above.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, may be obtained by any technique that providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableor hypervariable region derived from a murine mAb and a humanimmunoglobulin constant region.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-546) can be adapted to producedifferentially expressed gene-single chain antibodies. Single chainantibodies are formed by linking the heavy and light chain fragments ofthe Fv region via an amino acid bridge, resulting in a single chainpolypeptide.

Most preferably, techniques useful for the production of “humanizedantibodies” can be adapted to produce antibodies to the polypeptides,fragments, derivatives, and functional equivalents disclosed herein.Such techniques are disclosed in U.S. Patent Nos. 5,932, 448; 5,693,762;5,693,761; 5,585,089; 5,530,101; 5,910,771; 5,569,825; 5,625,126;5,633,425; 5,789,650; 5,545,580; 5,661,016; and 5,770,429, thedisclosures of all of which are incorporated by reference herein intheir entirety.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, such fragments include but are notlimited to: the F(ab′)₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

An antibody of the present invention can be preferably used in a methodfor the diagnosis of a condition associated with abnormal APP pathwayregulation and/or Alzheimer's Disease in a subject, or to identify asubject with a predisposition to said conditions, which comprises:measuring the amount of a polypeptide comprising the amino acid sequenceof any of the human homologs of the genetic modifiers identified inTable 1, preferably, the polypeptides of human homologs located onchromosome 10 disclosed herein, most preferably, the polypeptide encodedby SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO 53, i.e. the curated noc Asequences, or fragments thereof, in an appropriate tissue or cell from asubject wherein the presence of an elevated amount of said polypeptideor fragments thereof, relative to the amount of said polypeptide orfragments thereof in the respective tissue from a control subject isdiagnostic of said condition. Such a method forms a further embodimentof the present invention. Preferably, said detecting step comprisescontacting said appropriate tissue or cell with an antibody whichspecifically binds to a polypeptide that comprises the amino acidsequence of any one or more of the polypeptides discussed above or afragment thereof and detecting specific binding of said antibody with apolypeptide in said appropriate tissue or cell, wherein detection ofspecific binding to a polypeptide indicates the presence of any one ormore of said polypeptides or a fragment thereof.

Particularly preferred, for ease of detection, is the sandwich assay, ofwhich a number of variations exist, all of which are intended to beencompassed by the present invention.

For example, in a typical forward assay, unlabeled antibody isimmobilized on a solid substrate and the sample to be tested broughtinto contact with the bound molecule. After a suitable period ofincubation, for a period of time sufficient to allow formation of anantibody-antigen binary complex. At this point, a second antibody,labeled with a reporter molecule capable of inducing a detectablesignal, is then added and incubated, allowing time sufficient for theformation of a ternary complex of antibody-antigen-labeled antibody. Anyunreacted material is washed away, and the presence of the antigen isdetermined by observation of a signal, or may be quantitated bycomparing with a control sample containing known amounts of antigen.Variations on the forward assay include the simultaneous assay, in whichboth sample and antibody are added simultaneously to the bound antibody,or a reverse assay in which the labeled antibody and sample to be testedare first combined, incubated and added to the unlabeled surface boundantibody. These techniques are. well known to those skilled in the art,and the possibility of minor variations will be readily apparent. Asused herein, “sandwich assay” is intended to encompass all variations onthe basic two-site technique. For the immunoassays of the presentinvention, the only limiting factor is that the labeled antibody be anantibody that is specific for modifier polypeptide or a fragmentthereof.

The most commonly used reporter molecules in this type of assay areeither enzymes, fluorophore- or radionuclide-containing molecules. Inthe case of an enzyme immunoassay an enzyme is conjugated to the secondantibody, usually by means of glutaraldehyde or periodate. As will bereadily recognized, however, a wide variety of different ligationtechniques exist, which are well-known to the skilled artisan. Commonlyused enzymes include horseradish peroxidase, glucose oxidase,beta-galactosidase and alkaline phosphatase, among others. Thesubstrates to be used with the specific enzymes are generally chosen forthe production, upon hydrolysis by the corresponding enzyme, of adetectable color change. For example, p-nitrophenyl phosphate issuitable for use with alkaline phosphatase conjugates; for peroxidaseconjugates, 1,2-phenylenediamine or toluidine are commonly used. It isalso possible to employ fluorogenic substrates, which yield afluorescent product rather than the chromogenic substrates noted above.A solution containing the appropriate substrate is then added to thetertiary complex. The substrate reacts with the enzyme linked to thesecond antibody, giving a qualitative visual signal, which may befurther quantitated, usually spectrophotometrically, to give anevaluation of the amount of modifier protein which is present in theserum sample.

Alternately, fluorescent compounds, such as fluorescein and rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labeled antibody absorbs the light energy,inducing a state of excitability in the molecule, followed by emissionof the light at a characteristic longer wavelength. The emission appearsas a characteristic color visually detectable with a light microscope.Immunofluorescence and EIA techniques are both very well established inthe art and are particularly preferred for the present method. However,other reporter molecules, such as radioisotopes, chemiluminescent orbioluminescent molecules may also be employed. It will be readilyapparent to the skilled artisan how to vary the procedure to suit therequired use.

Polynucleotides encoding human homologs of genetic modifiers identifiedaccording to the methods of the present invention may be used in amethod to diagnose conditions associated with defects in the regulationof the APP pathway, including but not limited to Alzheimer's Disease orto identify individuals with a genetic predisposition to suchconditions. For example, said method comprises detecting the level oftranscription of mRNA transcribed from the gene encoding a human homologof a genetic modifier disclosed herein in an appropriate tissue or cellfrom a human, wherein abnormal transcription compared to control levelsis diagnostic of said condition or a predisposition to said condition.In particular, said genetic modifier comprises the nucleotide sequenceof any of the human homologs of the genetic modifiers identified inTable 1, preferably, the polypeptides of human homologs located onchromosome 10 disclosed herein, most preferably, the polypeptidesencoded by SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO 53, i.e. thecurated noc A sequences, or the polypeptide encoded by SEQ ID NO: 35,i.e. the EGR2 sequence, or the polypeptides encoded by SEQ ID NO: 41, orSEQ ID NO: 43, the ankyrin-related sequences.

Detection of a mutated form of a gene encoding a genetic modifieridentified according to the methods of the present invention which isassociated with a dysfunction will provide a diagnostic tool that canadd to, or define, a diagnosis of a disease, or susceptibility to adisease, which results from under-expression, over-expression or alteredspatial or temporal expression of the gene. Said diseases may include,but are not limited to, Alzheimer's Disease or other conditionscharacterized by errors in the regulation of the APP pathway.Individuals carrying mutations in said genes may be detected at the DNAlevel by a variety of techniques.

Nucleic acids, in particular mRNA, for diagnosis may be obtained from asubject's cells, such as from blood, urine, saliva, tissue biopsy orautopsy material. The genomic DNA may be used directly for detection ormay be amplified enzymatically by using PCR or other amplificationtechniques prior to analysis. RNA or cDNA may also be used in similarfashion. Deletions and insertions can be detected by a change in size ofthe amplified product in comparison to the normal genotype. Hybridizingamplified DNA to labeled nucleotide sequences encoding the human homologof a genetic modifier polypeptide of the present invention can identifypoint mutations. Perfectly matched sequences can be distinguished frommismatched duplexes by RNase digestion or by differences in meltingtemperatures. DNA sequence differences may also be detected byalterations in electrophoretic mobility of DNA fragments in gels, withor without denaturing agents, or by direct DNA sequencing (e.g., Myerset al., Science (1985) 230:1242). Sequence changes at specific locationsmay also be revealed by nuclease protection assays, such as RNase and SIprotection or the chemical cleavage method (see Cotton et al., Proc NatlAcad Sci USA (1985) 85: 4397-4401). In another embodiment, an array ofoligonucleotides probes comprising nucleotide sequence encoding agenetic modifier polypeptide of the present invention or fragments ofsuch a nucleotide sequence can be constructed to conduct efficientscreening of e.g., genetic mutations. Array technology methods are wellknown and have general applicability and can be used to address avariety of questions in molecular genetics including gene expression,genetic linkage, and genetic variability (see for example: M. Chee etal., Science, Vol 274, pp 610-613 (1996)).

The diagnostic assays offer a process for diagnosing or determining asusceptibility to disease through detection of mutation in a humanhomolog of a modifier gene by the methods described. In addition, suchdiseases may be diagnosed by methods comprising determining from asample derived from a subject an abnormally decreased or increased levelof polypeptide or mRNA. Decreased or increased expression can bemeasured at the RNA level using any of the methods well known in the artfor the quantitation of polynucleotides, such as, for example, nucleicacid amplification, for instance PCR, RT-PCR, RNase protection, Northernblotting and other hybridization methods. Assay techniques that can beused to determine levels of a protein, such as a polypeptide of thepresent invention, in a sample derived from a host are well known tothose of skill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

Such hybridization conditions may be highly stringent or less highlystringent, as described above. In instances wherein the nucleic acidmolecules are deoxyoligonucleotides (“oligos”), highly stringentconditions may refer, e.g., to washing in 6× SSC/0.05% sodiumpyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-baseoligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).Suitable ranges of such stringency conditions for nucleic acids ofvarying compositions are described in Krause and Aaronson (1991),Methods in Enzymology, 200:546-556 in addition to Maniatis et al., citedabove.

Thus in another aspect, the present invention relates to a diagnostickit which comprises:

(a) a polynucleotide of a human homolog of a genetic modifier identifiedaccording to the methods of the present invention, preferably, apolypeptide of a human homolog located on chromosome 10 disclosedherein, or a fragment thereof,

(b) a nucleotide sequence complementary to that of (a);

(c) a polypeptide of a genetic modifier of the present invention,preferably the polypeptide of a human homolog of the genetic modifiersidentified in Table 1, preferably, the polypeptide of a human homologlocated on chromosome 10 disclosed herein, or a fragment thereof; or

(d) an antibody to a genetic modifier polypeptide of the presentinvention, preferably to the polypeptide of a human homolog of thegenetic modifiers identified in Table 1, preferably, the polypeptide ofa human homolog located on chromosome 10 disclosed herein.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component. It is also contemplated that such akit may comprise components directed to one or more of said humanhomologs. Such a kit will be of use in diagnosing a disease orsusceptibility to a disease, particularly to a disease or conditionassociated with errors in the regulation of the APP pathway including,but not limited to, Alzheimer's Disease.

The nucleotide sequences of the human homologs of genetic modifiers ofthe present invention can also be used for genetic linkage analysis.Since the complete human genome sequence is known, the nucleotidesequence of interest can be specifically mapped to a particular locationon an individual human chromosome. The mapping of relevant sequences tochromosomes according to the present invention is an important firststep in correlating those sequences with gene associated disease. Once asequence has been mapped to a precise chromosomal location, the physicalposition of the sequence on the chromosome can be correlated withgenetic map data. Such data are found in, for example, V. McKusick,Mendelian Inheritance in Man (available on-line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes). Recent data indicate that there is a region on humanchromosome 10 linked to Alzheimer's Disease (Bertram et al.,Ertekin-Taner et al., Myers et al., Science 290, 2302-2305, 2000), thus,human homologs of genetic modifiers identified according to the methodsof endonucleolytic cleavage. Examples which may be used includeengineered hammerhead motif ribozyme molecules that can specifically andefficiently catalyze endonucleolytic cleavage of sequences encoding thegene products of Table 1.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Antisense molecules and ribozymes of the invention may be prepared byany method known in the art for the synthesis of nucleic acid molecules.These include techniques for chemically synthesizing oligonucleotidessuch as solid phase phosphoramidite chemical synthesis. Alternatively,RNA molecules may be generated by in vitro and in vivo transcription ofDNA sequences encoding the genes of Table 1. Such DNA sequences may beincorporated into a wide variety of vectors with suitable RNA polymerasepromoters such as T7 or SP6. Alternatively, these cDNA constructs thatsynthesize antisense RNA constitutively or inducibly can be introducedinto cell lines, cells, or tissues.

Vectors may be introduced into cells or tissues by many available means,and may be used in vivo, in vitro or ex vivo. For ex vivo therapy,vectors may be introduced into stem cells taken from the patient andclonally propagated for autologous transplant back into that samepatient. Delivery by transfection and by liposome injections may beachieved using methods which are well known in the art.

Gene specific inhibition of gene expression may also be achieved usingconventional double stranded RNA technologies. A description of suchtechnology may be found in WO 99/32619 which is hereby incorporated byreference in its entirety.

Still further, such molecules may be used as components of diagnosticmethods and kits whereby the presence of an allele causing diseasesassociated with abnormalities in the APP pathway and/or Alzheimer'sDisease may be detected.

Other objects, features, advantages and aspects of the present inventionwill become apparent to those of skill from the following description.It should be understood, however, that the following description and thespecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only. Various changes andmodifications within the spirit and scope of the disclosed inventionwill become readily apparent to those skilled in the art from readingthe following description and from reading the other parts of thepresent disclosure.

EXAMPLES

The following procedures are performed to conduct the examples:

Transgenic Flies

Methods for the creation of transgenic Drosophila melanogaster flies arewell known to one of skill in the art. Any conventional method can beemployed, for example, the basic laboratory techniques that are involvedin the creation of the flies of the present invention are described inSpradling, above. As contemplated herein, transgenic flies may becreated by direct fusion of DNA sequences of interest with expressioncontrol sequences as described below. For example, transformed strainsare generated using the constructs discussed above according toconventional methods. Several independent insertions may be obtained forthe constructs, UAS-Abeta40, UAS-Abeta42, UAS-C99wt, UAS-C99V7171(London mutation) and pGMR-Abeta42.

Fly Stocks

Gal4 lines that may be used to drive expression of the transgenes in thetransgenic flies of the present invention include, but are not limitedto, apterous-Gal4 and 7B-Gal4. Descriptions of the Gal4 lines mentionedand notes about their specific expression patterns can be found inFlybase (http://flybase.bio.indiana.edu). New transgenic strainsgenerated in house include strains carrying UAS Abeta₄₀ and Abeta₄₂, UASC99 wild type and UAS C99 London (carrying the London FADAlzheimer's-associated mutation) and GMR Abeta₄₂ transgenes.

The yw; BcElp/CyOHop strain, expressing transposase, and the strains yw;Gla/SM6a and yw, Dr/TM3 Sb Ser were obtained from R. Padgett, WaksmanInstitute, Rutgers University. w¹¹¹⁸ flies and GMR-GAL4 flies were fromthe Bloomington stock center. The pGMR-1 strain is a publicly availablestock and was obtained from G. Rubin's lab at UC Berkeley.

DNA Constructs and Molecular Techniques

A DNA fragment coding for the Aβ 42 peptide and fused to the humanpre-proenkephalin signal peptide is PCR amplified and cloned into theBgl II site of the Drosophila eye-specific P element transformationvector, pGMR (Hay et al., 1994 Development 120:2121-2129) and the insertis sequenced by automated fluorescence sequencing (ACGT Inc.). The humanpre-proenkephalin signal peptide has been shown to successfully drivesecretion of Aβ 42 from transfected mammalian cells. GMR is composed offive tandem copies of a response element derived from the rhodopsin- 1gene promoter, a binding site for the eye-specific transcription factorGLASS (Ellis et al., Development 119(3):855-65 (1993). Thus, Abetaexpression is driven in the pattern of the GLASS transcriptionalactivator in the eye. The above DNA fragment is subsequently cloned intoa P-element containing vector that facilitates the insertion of thetransgene into the Drosophila genome. All molecular manipulations aredone according to standard protocols. (See, for example, Sambrook,Fritsch and Maniatis, “Molecular Cloning A Laboratory Manual, 2^(nd)Edition, Cold Spring Harbor Laboratory Press, 1989).

A DNA fragment coding for the C99 peptide and fused to the humanpre-proenkephalin signal peptide is PCR amplified and cloned into thepUAST transformation vector as described in Brand and Perimmon.

The constructs UAS-Abeta40, UAS-Abeta42, UAS-C99wt and UAS-C99V7171contain the pre-proenkephaline gene signal peptide followed by fragmentsof human Abeta (40 or 42) or C99 (wild type or with the Londonmutation). The human fragments are cloned into the pUAST vector asdescribed in Brand and Perrimon, above. Cloning into this vector placesthe UAS sequence upstream of the transcribed region of the inserted geneand also allows integration into the fly genome through P-elementrecombination.

Genetic Crosses, Analysis and Visualization of Phenotypes

Flies are crossed according to conventional methods except that allcrosses are kept at 29° C. for maximal expression of phenotypes. In thebinary Gal4 expression system, this temperature maximizes activity ofthe Gal4 protein. In the case of pGMR-Abeta42, it is observed that thephenotype is stronger at 29° C., so these flies are kept at thistemperature as well.

Western Analysis

Ectopic gene expression can be assayed by performing Western analysisaccording to conventional methods. Antibodies that may be used includethe human 6E10 monoclonal antibody raised against the beta amyloidportion of the APP gene and which also recognizes the C99 portion of APP(Senetek PLC, Napa, Calif.).

Western Protocol

To detect expression of the Aβ 42 peptide, flies of genotypesK18.1/K18.1, K18.3/K18.3, K18. 1/K18.1; K18.3/K18.3, KJ103/TM3Sb Ser,KJ103/KJ103, KJ54/CyO;KJ54/TM2 Ubx and pGMR-1 (flies carrying pGMRvector without insert) are reared at 29° C. 80-90 Drosophila heads fromeach of the above strains are collected, placed in an eppendorf tube ondry ice containing 100μl of 2% SDS, 30% sucrose, 0.718 M Bistris, 0.318M and Bicine, with “Complete” protease inhibitors (Boehringer Mannheim)and are ground using a mechanical homogenizer. Samples are heated for 5min at 95° C., spun down for 5 min at 12, 000 rpm, and supernatants aretransferred into a fresh eppendorf tube. 5% β-mercaptoethanol and 0.01%bromphenol blue are added and samples are boiled prior to loading.Approximately 200 ng of total protein extract is loaded for each sample,on a 15% Tricine/Tris SDS PAGE gel containing 8M Urea. The Aβ 1-42peptide control is human β-amyloid [1-42] (BIOSOURCE International, #03-111). Samples are run at 40V in the stacking gel, and at 120V in theseparating gel. Samples are transferred to PVDF membranes (BIO-RAD, #162-0174) for 1 hr @100V, and the membranes are subsequently boiled inPBS for 3 min. Antibody hybridization is as follows: the primary Ab 6E10(SENETECK PLC, # 300-02), which recognizes the first 19 amino acids ofthe Aβ peptide, is used for probing (at a concentration of 1:2000) in 5%non-fat milk, 1×PBS containing 0.1% Tween 20, for 90 min @ RT. Samplesare washed 3 times for 5 min., 15 min. and 15 min. each, in 1×PBS-0.1%Tween-20. The secondary Ab is anti-mouse-HRP (Amersham PharmaciaBiotech, # NA 931) and is used at 1:2000 in 5% non-fat milk, 1×PBScontaining 0.1% Tween 20, for 90 min at RT. Samples are washed 3 timesfor 5 min., 15 min. and 15 min. each, in 1×PBS-0.1% Tween-20. ECL (ECLWestern Blotting Detection Reagents, Amersham Pharmacia Biotech, # RPN2209) is used for detection. Histology Plastic sections of fly heads areperformed according to conventional methods, for example, as accordingto the protocols described in: Drosophila Protocols, page 236. Eds. W.Sullivan, M. Ashburner, S. Hawley, CSHL Press 2000

Cryosections

Adult eyes are cryosectioned according to Wolff, in DrosophilaProtocols, CSHL Press, 2000, sections 13.1 and 13.2. The primaryantibody is the monoclonal 6E10 (Senetek), recognizing the human Aβ 42peptide, used at a dilution of 1:3000. The detection system is theVectastain ABC Kit (with biotinylated anti-mouse IgG secondary, andHorseradish peroxidase H) (Vector Laboratories). The followingmodifications are made to the protocol by Wolff: prior to incubationwith the 6E10 primary antibody, cryosections are blocked in blockingsolution containing normal horse serum, according to the Vectastain ABCKit protocol. Incubation with the secondary (preadsorbed with pGMR-1 eyetissue) is done in PBS/1% BSA containing 1-2% normal horse serum, alsoaccording to the Vectastain ABC Kit protocol. The procedure for the ABCKit is followed; incubations with the ABC reagent are done in PBS/0. 1%saponin, followed by 4×10 min. washes in PBS/0.1% saponin. Sections arethen incubated in 0.5 ml per slide of the Horseradish Peroxidase Hsubstrate solution, 400μg/ml 3,3′diaminobenzidene (DAB), 0.006% H₂O₂ inPBS/0.1% saponin, and the reaction is stopped after 3 min. with 0.02%sodium azide in PBS. Sections are rinsed several times in PBS anddehydrated through an ethanol series before mounting in DPX (Fluka).

RNA Profile Characterization for Compound Screening

RNA profiles may be assayed according to known methodology, includinguse of traditional Northern blot analysis as well as microarray chiptechnology (Incyte Pharmaceuticals, Palo Alto, Calif.; Affymetrix ,Santa Clara, Calif.).

Example 1

The Rough Eye Phenotype Induced by Ectopic Expression of Aβ 42

In order to elucidate the largely unknown pathways and mechanism(s) bywhich Aβ 42 causes neurodegeneration, the Drosophila eye, a neuraltissue, is used as a model. In an effort to mimic the disease-specificAβ 42 overexpression, transgenic flies whose genome comprises theGMR-Abeta42 amyloid transgene are created using the GMR fusionexpression system disclosed above in order to ectopically express thetransgene in the developing Drosophila eye.

I. Aβ 42 Overexpression Causes Rough Eye Phenotypes

In order to express the Aβ 42 peptide in the Drosophila eye, the Aβ 42sequence is cloned into the pGMR vector. The pGMR (Glass MultimerReporter) vector contains a pentamer of truncated binding sites for theGlass transcription factor. Glass is expressed widely during eyedevelopment, starting in the eye discs, the precursors of adultDrosophila eyes, where it is detected in differentiating photoreceptorneurons. It continues being expressed specifically in the eye duringpupal and adult development (Moses et al, 1989 Nature 340(6234):531-536; Moses and Rubin, 1991 Genes Dev. 5:583-593). GMR-elementexpression in 2 week-old flies is examined using a reporter gene andgood expression detected, suggesting that GMR element is active wellinto adulthood. Thus, GMR-regulated expression is directed to the eyetissue throughout the development of the eye, as well as duringadulthood, making it a suitable system for expression of AP 42.

Two independent transgenic lines are originally established with thepGMR-Aβ 42 construct, K18.1 and K18.3. In addition, another transgenicline, pGMR-1 expressing the same vector without an insert, is examinedas a negative control. Control flies with the pGMR-1 transgene and fliescarrying one copy of either the K18.3 or the KJ18.1 transgene do notshow a rough eye. Similarly, flies carrying one copy each of both of theabove transgenes (K18.3 and K18.1) or two copies of the K18.1 transgenealso have wild type eyes. In contrast, flies with two copies of K18.3have a mild rough eye phenotype; examination of fly eyes under lightmicroscopy indicate that ectopic overexpression of Aβ 42 disrupts theregular trapezoidal arrangement of the photoreceptor cells of theommatidia (identical single units, forming the Drosophila compound eye.The above observations suggest that there might be a dose response ofthe rough eye phenotype to the copy number of transgenes present in thefly genome. To further examine this hypothesis, the number of transgenesis increased to three (2 copies of K18.1 with one copy of K18.3 or onecopy of K18.1 with two copies of KJ18.3). These strains also showedrough eyes. Finally, when four copies of the transgene were present (2copies of K18.1 with 2 copies of K18.3), flies showed a much more severerough eye phenotype confirming the dose response hypothesis. Thepenetrance of the rough eye phenotypes is 100% in all geneticcombinations. It must also be noted that a more severe phenotype isobserved when flies are reared at 29° C. A temperature requirement forexpressivity of eye phenotypes has been described previously (Karim andRubin, 1998 Development 125(1):1-9) and may be specific for the eye,even though it is not restricted to GMR-containing expression systems.Such dependence could be attributed to higher transcriptional and/ormetabolic rates, or altered protein conformation at the highertemperature.

II. Aβ 42 Transgenics Display Rough Eye Phenotypes, the Severity ofWhich Depends on Transgene Copy Number.

It is well established that the expression level of transgenes inDrosophila depends on the chromosomal location of the specificinsertions, a phenomenon known as “position effect” (Kellum, R. andSchedl, P. (1991). Cell. 64: 941-50). It is possible then, by generatingadditional independent insertions with the same transgene, to recovertransgenic lines that express different levels of the transgenicprotein. Thus, it might be possible to isolate transgenic lines thatwould express the Aβ 42 transgene at a high enough level to cause aphenotype at a lower temperature (25° C.), thus reflecting morephysiological conditions. To test this hypothesis, new insertions of thepGMR-Aβ 42 transgene in the fly genome are generated, using “P-elementhopping” (Robertson, H. M. et al. (1988). Genetics 118: 461-470).

A total of 19 independent lines of the pGMR-Aβ 42 construct in newchromosomal locations are established. The new strains are judged ascarrying new insertions based on the chromosomal linkage or homozygouslethal condition of the transgene, as well as by differences in eyecolor (caused by differential expression levels of the white gene, usedas a transformation marker). Young larvae of the above new strains aresubsequently raised at 29° C. until eclosion and examined for thepresence of an eye phenotype. Of the 19 new lines, 7 lines, or 38%, showa rough eye phenotype. The strains that display a rough eye phenotypeare subsequently raised at 25° C. and scored for an eye phenotype.

The new transgenic lines show varying degrees of phenotypic severity,some of them displaying a more severe phenotype than what was originallyobserved in the K18.1 and K18.3 line. One such example is the KJ.103line, in which one copy of the transgene renders the adult eyes mildlyrough, characterized by the presence of interspersed darker “spots”(corresponding to deeper-red pigmented ommatidia) on the ventral side ofthe eye, while two copies of the transgene cause extensivedisorganization of photoreceptors. More importantly, this specific linedisplays the rough eye phenotype even when the flies are raised at 25°C. When KJ.103 flies are raised at 29° C., the severity of phenotypecaused by either one or two copies of the transgene is increaseddramatically.

In summary, rough eye phenotypes caused by the Aβ 42 peptide show arange of severity. The very mild lines typically display numerousdark/black “spots” on the ventral side of the eye, while mild lines havea more rough, disorganized appearance covering the ventral portion ofthe eye. Moderate lines show greater roughness over the entire eye,while in more severe lines the entire eye seems to have lost/fused manyof the ommatidia and interommatidial bristles, and the entire eye has asmooth, glossy appearance. Interestingly, the size of the eye is onlymoderately affected in flies with the highest level of the Aβ 42expression (strain KJ54). This is consistent with observations in fliesexpressing human oc-synuclein (Feany, M. B. and Bender, W. W. (2000)Nature 404:394-398. In flies expressing poly-glutamine expanded humanhuntingtin, a very slight reduction of eye size is observed, in thestrongest-expressing transgenic lines (Warrick, J. M., et al (1998).Cell 93: 939-949). The above results suggest that neurodegenerationinduced by over-expression of human disease genes differs from thephenotypes caused by overexpression of genes acting in apoptoticpathways (Grether, M. E. et al. (1995). Genes Dev. 9, 1694-1708), inwhich the size of the eye is primarily affected.

Based on the above results, it is hypothesized that the severity of therough eye phenotype depends on the amount of Abeta protein present. As aconsequence of this hypothesis, it should be expected that the KJ. 103transgene displays a higher level of protein expression than the K18.3transgene (see below).

III. Expressivity of the Rough Eye Phenotype Correlates with β 42Protein Levels

To determine if the severity of the rough eye phenotype correlates withexpression levels of the Aβ 42 peptide, Western blot analysis of proteinextracts from Drosophila heads are performed (strains used are describedin methods above). Results indicate that animals with two copies of thetransgene have roughly twice the amount of Aβ 42 peptide than animalswith one transgene copy. Interestingly, even though flies with twocopies of K18.1 express the Aβ 42 peptide in detectable quantities, theyhave no visible adult eye phenotype. Flies with two copies of thehigher-expressing K18.3 transgene, expressing overall larger quantitiesof Aβ 42 peptide do show the rough eye phenotype. This is also true forflies expressing two copies of the K18.1 and two copies of the K18.3transgenes. Flies expressing only one copy of the KJ103 transgene haveroughly equal amounts of protein as flies expressing two copies of theK18.3 transgene, confirming the hypothesis that the KJ103 transgeneshows higher levels of relative protein expression.

The above results indicate that there is a requirement for a certainlevel of Aβ 42 protein in order to generate a visible phenotype. It isstill possible that lower amounts of Aβ 42 expression cause minordisruptions that would only be visible at the ultrastructural level. Totest this, thin sections (1.5μm) from adult fly heads are examined.These data indicate that, compared to eyes from a fly carrying the emptypGMR vector, in which the tolouidine-blue staining photoreceptors areregularly arrayed, flies carrying one copy of the moderately expressingK18.3 transgene have small abnormalities-some photoreceptors aremissing, blue-staining masses are forming around the ommatidia and somegaps are appearing in the tissue. These eyes appear normalmacroscopically.

Sections from eyes expressing two copies of the K18.3 transgene, inagreement with observations at the macroscopic level, display variabledisorganization. As the phenotype gets worse, the concentration ofdense, staining masses around the ommatidia increases, as do the gaps inthe tissue. The ommatidia look smaller and are missing photoreceptors.Two copies of the higher expressing KJ103 transgene show a phenotypesimilar in severity. Finally, eyes from Drosophila expressing fourcopies of the strong expressing KJ54 transgene show an almost completeloss of photoreceptors. Additionally, these eyes show an abundance ofdense, staining masses and of tissue gaps. Even though it is not clearat this point whether the dense, staining masses that surround theommatidia are abnormal/dying cells or whether they contain aggregatingAbeta peptide, it is clear that their accumulation is coincident withobserved overall eye degeneration.

In order to visualize the expression of beta-amyloid on the eye tissue,sections of Aβ expressing eyes are stained with an antibody recognizingthe human Aβ peptide. Transverse sections of eye tissue show a punctatestaining that is absent in controls. It is hypothesized that thispunctate staining corresponds to small aggregates/deposits of betaamyloid. Cellular localization of this staining as well as the exactnature of the aggregate/deposit, using known Aβ staining dyes is underinvestigation.

In summary, it is disclosed herein that introduction of more copies ofthe Aβ 42 transgene in the Drosophila eye, reflected by increased levelsof Aβ protein, has an additive affect on the rough eye phenotype. It ispossible that a certain concentration of the Aβ 42 peptide is needed toaffect its aggregation/conformation state. Alternatively, saturatinglevels of the peptide might be needed for manifestation of the toxiceffect. The fact that Aβ exerts neurotoxic effects in several signalingpathways, (intracellular calcium levels, oxidative stress, inflammatoryresponse, muscarinic and nicotinic receptor signaling, reviewed inFraser, S. P., et al (1997). Trends Neurosci. 20: 67-72; Mattson, M. P.(1997). Physiol. Rev. 77: 1081-1132; and Coughlan, C. M. and Breen, K.C. (2000). Pharmacol. and Ther. 86: 111-144; Hellstrom-Lindahl andCourt, 2000 Behav Brain Res. 113 (1-2):159-168), might indicate the needfor saturating levels in order to cause disruptions. It is clearhowever, that expressing moderate amounts of the peptide seem to have noconsequence for the structure of the adult eye at the grossmorphological level.

IV. Rough Eye Phenotype Induced by Aβ 42 Peptide Worsens with Age

It is well established that in Alzheimer's patients, chronicaccumulation of Aβ peptide leads to initial manifestation of the diseaseand to progressive worsening of the symptoms. In order to test whetherone could mimic this aspect of the disease in the Drosophila model, thedegree of roughness of the eye phenotype in aged flies is recorded.

Two strains of flies, expressing pGMR1 (as a negative control) and K18.3are examined. K18.3 flies are used because in this transgenic strainthere is a range of phenotypic severity and thus it is easier to recordchanges. Flies from the two strains are raised at 25° C. and 0-2daysafter eclosion they are transferred to 29° C., to induce higherexpression of the transgene. Flies are scored for eye phenotypeapproximately every week, for a total of one month, thereafter. TheK18.3 flies are classified into three different groups (moderate, mild,intermediate), according to the observed severity of the eye phenotype.As mentioned previously, pGMR1 expressing flies did not show any eyephenotype.

Data indicates a shift in the phenotypic severity of the Abetaexpressing flies as they age: when flies first eclose, no eyes with anintermediate phenotype are observed, whereas 15% of the population atseven days has an intermediate phenotype. Also by seven days, all of theprogeny show a degree of rough eye phenotype, whereas 42% do not showany phenotype upon eclosion. By 32 days, even though a large number offlies have died, the overall ratio of flies with mild versusintermediate phenotype is not significantly changed, suggesting that themaximum effect of Abeta expression has been reached.

The Drosophila model disclosed herein appears to be mimicking theprogressive and age-associated worsening of the Alzheimer's diseasesymptoms, an important aspect of the disease. The observed increase inthe severity of the eye phenotype as flies age could be attributed toincreased sensitivity of neuronal cells to the levels of Aβ peptide.Indeed, as mentioned above, Aβ peptide is being produced throughout theadult stage of Drosophila. It is thus possible that increased levels ofAβ cannot be effectively turned over, resulting in accumulation of thepeptide in the Drosophila cells. Alternatively, it is possible that agedcells are more vulnerable to the presence of Aβ peptide.

V. The Rough Eye Phenotype and the Degree of Apoptotic Cells in LarvalEye Imaginal Discs and Adult Eyes

As mentioned earlier, the Aβ 42 peptide has known toxic effects and itis suggested that it plays a role in apoptosis. Based on this, thirdinstar larval eye imaginal discs, the precursors of the adult eye, areexamined for evidence of apoptosis, or programmed cell death. Dissectedeye imaginal discs from K18.1/K18.1; K18.3/K18.3 larvae, raised at 29°C., are stained with acridine orange according to conventional methods,which causes fluorescence of apoptotic cells. As controls, the followingstrains, none of which shows any eye abnormalities, are used: w¹¹¹⁸ (awild-type control) and pGMR-1 (carrying the “empty” pGMR vector) grownat 29° C. and GMR-GAL4 (expressing Gal4 under the control of the GMRelement), raised at 18° C.

Results indicate that little or no cell death is seen in the wild-typecontrol, w¹¹¹⁸ . In contrast, some amount of cell death can be detectedin the K18.1/K18.1; K18.3/K18.3 line. When the controls that carry thepGMR vector but do not display any eye phenotype (pGMR-1 and GMR-GAL4),are examined, some cell death is also observed, comparable in extent tothat observed in the experimental flies, K18.1/K8.1; K18.3/K18.3.Therefore, it seems likely that a certain amount of cell death istolerated during eye development and does not cause any adult eyedefects, at least at the gross morphological level. In addition, it issuggested herein that if apoptosis has any involvement in the generationof the rough eye phenotype, it is not manifested during the earlydevelopment of the eye.

To test whether the observed rough eye phenotype is caused by apoptosisduring the adult stages of Drosophila, the apoptosis inhibitor DIAP1 isco-expressed in the Drosophila eye. Co-expression of DIAP1 in eyesexpressing Abeta would be expected to suppress, at least partially, therough eye phenotype (data not shown). Since no suppression with twodifferent DIAP-expressing strains is observed, it may be that theobserved rough eye phenotype is not caused by ectopically inducedapoptotic cell death. The same results were obtained when theantiapoptotic baculoviral P35 gene was used. These results suggest thatthe effects caused by the Aβ 42 peptide in the Drosophila eye might bemediated by cellular pathways that do not result in apoptosis.

The actions of Aβ 42 are quite complex and could affect other proteinsknown to be factors in AD development. It has been shown that PS 1 andPS 2 co-immunoprecipitate with APP (Xia et al., 1997 PNAS USA 94(15):8208-13) and that Aβ 42 can directly bind PS 2 in vitro (Czech etal., 1999 Society for Neuroscience 25:641.1). It is interesting to notethat overexpression of wild type and mutant PS forms also results inenhanced susceptibility to apoptosis in several experimental systems,including the Drosophila eye (Ye, Y. and Fortini, M. (1999). J. CellBiol. 146: 1351-1364). In these studies, it is suggested that Drosophilapresenilin (Dps) exerts a dominant negative effect when expressed athigh levels. It is unclear how Dps causes apoptosis of cells, but themechanism could involve the dysregulation of the Notch and/or Wntdevelopmental signaling pathways (reviewed in Anderton et al., 2000 Mol.Med. Today 6:54-59). It is unclear whether Aβ 42 overexpression in thesystem disclosed herein could be affecting Dps function by possiblyinterfering with one or more of these signaling pathways. Interestingly,overexpression of Aβ 40 or Aβ 42 enhances a Dps (Drosophila presenilin)induced phenotype in the same tissue (data not shown), suggestinginvolvement of the two proteins in the same pathway.

Example 2

Concave Wing Phenotype Induced by Ectopic Expression of C99

Transgenic Drosophila that carry a copy of pUAS-C99 (either wild type orwith the London mutation ) and a copy of apterous-Gal4 are created usingstandard methods and as discussed above. Data indicate that these fliesexhibit a malformation of their wings in that the wing blade is curvedin a concave manner. These effects are confirmed with multipleindependent insertions of the C99 transgene. Western analysis confirmsexpression of this transgene. Protein extracts from whole larvaeexpressing the C99 (either wild type or with the London mutation) underthe control of daughterless-Gal4 (a ubiquitously expressed Gal4 driver)show a protein band of the expected size for C99, which immunoreactswith the 6E10 antibody (raised against the first 16 amino-acids of C99).Data exists that when the portion of the human APP gene referred to asC100 was inserted into the genome of Drosophila and expressed in thewing disc, it did not generate any visible phenotype (Fossgfreen et al.,PNAS 95:13703-13708 (1998)). In contrast, data reported herein indicatethat flies transgenic with the equivalent C100 region of human APP(called here C99), fused to a different signal peptide, display a wingmalformation.

Example 3

Cognitive Defects Induced by Ectopic Expression of C99

Transgenic Drosophila that carry a copy of UAS-C99 (either wild type orwith the London mutation) and a copy of 7B-Gal4 (which allows expressionin the mushroom body of the brain) are created using standardtechniques. Cognitive defects of these flies can be examined byconducting olfactory, locomotion or learning and memory assays accordingto conventional methods. For example, altered locomotory behavior isobserved in the above flies, tested using the “dark reactivity” set-up,described by Benzer, S. PNAS 58:1112-1119 (1967). Specifically, fliescontaining a copy of UAS-C99 and a copy of 7B-Gal4 do not respond tomechanical agitation as well as wild type flies, walking less quicklythan wild type flies after being tapped to the bottom of the assayapparatus. The “dark reactivity” test for locomotion is also describedin “Behaviour, Learning and Memory” In: Drosophila, A PracticalApproach. Ed. D. B. Roberts (1998) Oxford University Press Inc. New Yorkpage 273.

Example 4

Genes that Modify Drosophila Phenotypes as Targets for Alzheimer DiseaseTherapeutics

As disclosed in detail below, genetic screens were set up in order toidentify genetic modifiers of the concave wing phenotype described inExample 2. Candidate modifiers tested included known modifiers of twoDrosophila phenotypes induced by ectopic expression of Drosophilapresenilin (Dps) in the wing and scutellum (G. Boulianne, Hospital forSick Children, Toronto, Canada, personal communication), as well asmutations in other candidate genes. Based on the recent discovery of achromosome 10 AD gene “hot spot”, chromosomal mapping of the humanhomologs of the above mentioned Drosophila genetic modifiers wasperformed. Data disclosed below indicate that the human homologs ofseveral of the genetic modifiers disclosed herein are also located onchromosome 10 and it is contemplated herein that these genes arerelevant targets for the development of pharmaceuticals useful for thetreatment of Alzheimer's Disease as well as other conditions associatedwith errors in the regulation of the APP pathway.

A total of 93 mutations were screened in order to identify geneticmodifiers of the concave wing phenotype induced by ectopic expression ofC99 in the wing of Drosophila. The screen is based on measuring thechange in the penetrance of the wing phenotype when the externalmutation is present. More specifically, the number of flies with mutantwings compared to the number of flies with wild type wings are countedin the experimental group (flies expressing both C99 and mutation beingtested) and the control group (flies expressing only C99). Thesignificance of the change in penetrance of the mutant phenotype isevaluated by measuring the P value by a T test, in the above mentionedfour groups. Mutations were considered to significantly modify the C99phenotype when P<0.05.

A list of genetic modifiers that affect the C99-overexpression phenotypeand the Drosophila genes associated with these genetic modifiers areprovided in Table 1.

All of the mutations identified as modifiers of presenilin and C99overexpression phenotypes were insertional mutations (mediated byinsertion into the Drosophila genome of the P-retroviral liketransposable element). The exact chromosomal location of each of theseinsertions has been previously determined (Drosophila Genome ProjectBDGP, http://www.fruitfly.org). In order to identify the transcript(s)affected by each of these insertions, we scanned a 10 kB genomic area tothe right and to the left of each insertion for known or predictedDrosophila transcripts. The following criteria were adopted forselection of the most likely transcript affected by a given insertion:

a) distance of transcripts from the site of insertion, and b)orientation of a transcript relative to the insertion.

If a genomic area contained more than one candidate transcripts with thesame orientation as the insertion, all those closest to the insertionwere selected for further analysis. The translated protein sequences ofthe Drosophila transcripts from the above analysis form “Set A”.

The presence of human homologs of the above Drosophila proteins (in “SetA”) in an AD-linked area of human chromosome 10 was examined. Twocandidate regions around Sequence Tagged Site (STS) markers on humanchromosome 10 have been identified (Bertram et al., Ertekin-Taner etal., Myers et al., Science 290, 2302-2305, 2000) by linkage analysis. Wemapped STS marker sequences used in these linkage analysis studies orSTS sequences adjacent to these markers to the Celera genome data byblastn (Altschul et al., 1997) sequence comparisons. Based on thismapping information a subset of human chromosome 10 was defined thatincluded the two candidate regions showing significant linkage (Bertramet al., 2000; Ertekin-Taner et al, 2000; Myers et al., 2000) and theregion in between. The DNA sequence (Celera contigs) and thecorresponding list of Celera protein translations were retrieved for thesubset defined and put into blast format databases. The DNA sequence andthe list of Celera protein translations for the above described genomicregions form “Set B” and “Set C”, respectively.

A tblastn search with “Set A” against “Set B” and a blastp search with“Set A” against “Set C” were then performed. Initially tblastn andblastp hits with E-values lower than 10⁻⁵ were selected. Then the bestmatch of each fly protein from these searches was chosen and thecorresponding fly genes/transcripts were checked for their associationwith genetic modifiers of the Dps and C99 phenotype. The resultingfourteen pairs of fly and human transcripts/proteins form “Set D”.

The fourteen protein pairs in “Set D” were tested for stringent orputative orthology. This was accomplished by blastp comparisons to acombined database of all human and all fly Celera proteins. First, thefly proteins in “Set D” were compared to each protein in this database.Then the resulting best human matches for each of the fly proteins wereagain compared to the combined human/fly protein database. A human matchwas classified as a stringent ortholog if all of the following fourcriteria were fulfilled:

-   1. fly protein X has best match with human protein Y-   2. fly protein X does not have a better match with another fly    protein than with human protein Y-   3. human protein Y has best match with fly protein X-   4. human protein Y has no better match with another human protein    than with fly protein X

If only criteria 1) and 3) are fulfilled, a human match is classified asa putative ortholog regardless of whether fly protein X had a bettermatch with another fly protein, human protein Y had a better match withanother human protein or both. All other human matches are deemedhomologs. After the orthology test, candidate human genes areprioritized according to the following:

-   a) human gene is homolog of fly gene affected by Drosophila genetic    modifier, identified in genetic screen-   b) degree of sequence similarity of the human protein (encoded by    the human gene in a) to the Drosophila protein (encoded by the    Drosophila gene in a)-   c) human protein is stringent or putative ortholog of fly protein-   d) chromosomal location of the human gene with respect to STS    markers, other candidate genes or known AD genes-   e) putative function of the human protein and/or the homologous    Drosophila protein-   f) evidence that the predicted human gene is expressed-   g) existence of validated or predicted coding and/or non-coding SNPs    in the coding region of the human gene.

To check whether the human homolog gene is expressed, the Incyte LifeSeqEST database was searched with the corresponding predicted humantranscripts identified from the Celera database using blastn.

Based on these criteria, it is contemplated herein that 4 differenthuman genes are AD related genes located on human chromosome 10. Beloware listed the putative proteins, encoded by these proposed human genes.

-   -   (1) hCP50765 (EGR2) SEQ ID NO: 35    -   (2) hCP41313 (homologous to the fly gene nocA) SEQ ID: 15, SEQ        ID NO: 17 or SEQ ID NO 53    -   (3) hCP33787 (ankyrin-related protein) SEQ ID NO: 41    -   (4) hCP51594 (ankyrin-3) SEQ ID NO: 43

The Celera predicted transcript hCT15097 was manually curated to producetwo putative forms of the protein hCP41313. Curation was performed byidentifying ESTs corresponding to this locus by blastn searches of theIncyte LifeSeq and public EST databases and aligning the identified ESTswith the Celera predicted transcript sequence. The curation producedslight changes in the C-terminal amino-acid sequence and putativeadditional residues at the N-terrninus of the predicted amino-acidsequence. The changes in the C-terminal part of the human proteinsequence lead to an improved alignment with the fly nocA in this region.Because of the additional residues at the N-terminus Met 64 and Met 100in the Celera protein sequence (hCP41313) correspond to Met 114 and Met150 in the translation of the complete curated nocA a homolog transcriptsequence respectively. We have subsequently analyzed and compared cDNAsequences from the Novartis FGA cDNA collection and the Incyte cDNAcollection. Based on these analyses we have cloned and sequenced a cDNAclone corresponding to the human nocA gene on chromosome 10. The 5′ endof this cDNA clone consists of cDNA obtained from proprietary Novartisclone fga94341 and the 3′ end of this clone consists of cDNA obtainedfrom Incyte clone 242278.1 (SEQ ID NO: 52, SEQ ID NO. 53).

EGR2 is a putative ortholog of Celera predicted fly transcript CT23724(see Table 1), which corresponds to the Drosophila stripe gene. The flymutation P1505 (see Table 1), which affects the stripe gene, modifiesonly the presenilin phenotype.

Human nocA is a putative ortholog of Celera predicted fly transcriptCT14619 (see Table 1), which corresponds to the Drosophila nocA gene.The fly mutation EP2173 (see Table 1), which affects the nocA gene,modifies both the presenilin and C99 overexpression phenotypes.

EGR2 is a C2H2 type zinc finger transcription factor regulating PNSmyelination. In mice it has been shown to be important for hindbraindevelopment (Schneider-Maunoury et al., Cell 75, 1199-1214, 1993;Swiatek & Gridley, Genes Dev. 7, 2071-2084, 1993). Four mutations inEGR2 have been described to be associated with inherited peripheralneuropathies (Warner et al., Nature Genet 18, 382-384, 1998; Timmermanet al., Neurology 52, 1827-1832, 1999).

The nocA human homologue is a putative transcription factor with a C2H2type zinc finger domain. While its exact function has yet to bedetermined, according to data disclosed herein, it may play asignificant role in the pathology of Alzheimer's Disease. The Drosophilaprotein encoded by the nocA gene is a transcription factor involved inthe development of the embryonic brain and the adult ocellar structures.

Ankyrin-3 exists in two brain specific isoforms of 480 and 270 kDa(Kordeli et al., J Biol Chem 270, 2352-2359, 1995). Neural-specificAnkyrin-3 polypeptides are candidates to participate in the maintenanceand targeting of ion channels and cell adhesion molecules to nodes ofRanvier and axonal initial segments. Ankyrin-3 has been shown toassociate with the voltage dependent sodium channel in vitro and toco-localize with this molecule at nodes of Ranvier, axonal initialsegments, and the neuromuscular junction (Srinivasan et al., Nature 333,177-180, 1988; Kordeli et al., J Cell Biol 110, 1341-1352, 1990; Kordeli& Bennett, J Cell Biol 114, 1243-1259, 1991; Flucher & Daniles, Neuron3, 163-175, 1989).

The second human homologue of fly transcript CT18415 belongs to thefamily of ankyrin-related proteins (hCP33787). The corresponding gene islocated 469 kbp from insulin-degrading enzyme (IDE). In addition toankyrin-repeats, hCP33787 contains a sterile alpha motif (SAM) domain.The SAM domain has been suggested to be involved in the regulation ofdevelopmental processes (Shultz et al., Protein Sci 6, 249-253, 1997),has been described as mediating specific protein-protein interactions,and has been suggested to form extended polymeric structures (Thanos etal, Science 283, 833-836). The SAM domain is included in the alignmentbetween fly transcript CT 18415 and hCP33787. We speculate that it mightplay a role in the aggregation of β-amyloid.

It has been hypothesized that γ-synuclein might be involved in AD(Luedecking et al., Neuroscience Letters 261, 186-188, 1999). Wepostulate that γ-synuclein might interact with the ankyrinrepeat-containing protein hCP33787. In support of this, aprotein-protein interaction between synphilin, an ankyrin repeat-relatedprotein, and α-synuclein has been shown (Engelender et al., Nature Gen22, 110-114, 1999). It is also known that members of the synucleinfamily share a high degree of sequence similarity (64% sequence identitybetween α-synuclein and γ-synuclein, Lavedan, Genome Res 8, 871-880,1998). Since the fold of an ankyrin-repeat unit is conserved, the abovearguments add support to a putative protein-protein interaction betweenhCP33783 or hCP51594 and y-synuclein. It is of interest to note that acoding SNP (E→K) is predicted for hCP33787 at sequence position 48,which corresponds to the start of the ankyrin-repeat region. Thissequence variation could be relevant in the context of a putativeγ-synuclein-ankyrin interaction because it involves oppositely chargedamino acid side chains. The predicted SNP in the ankyrin-related proteinis particularly interesting as γ-synuclein has a validated coding SNP(V→E) at position 110 (Ninkina et al., Hum Mol Genet 7, 1417-1424,1998), that codes for the exchange of a neutral by a negatively chargedamino acid side chain. We postulate that these polymorphisms might berelevant to a putative interaction of γ-synuclein with either hCP33783or hCP51594. TABLE 1 Genetic modifiers modifier flyCT Start End hCG hCThCP Start End E-value EP(2)2107 CT25384 94 183 hCG37225 hCT28457hCP47994 96 185 4.00E−32 EP(2)2122 CT11970 54 411 hCG22190 hCT13283hCP39677 12 348 4.00E−75 EP(2)2151 CT3996 27 392 hCG22926 hCT14025hCP40373 39 415 1.00E−109 EP(2)2162 CT7676 15 374 hCG30594 hCT21765hCP44907 13 373 3.00E−97 EP(2)2173 CT14619 10 531 hCG23983 hCT15097hCP41313 100 564 1.00E−25 EP(2)2205 CT9828 93 619 hCG41821 hCT33094hCP51674 668 1180 4.00E−66 EP(2)2511 CT11457 6 258 hCG20663 hCT11743hCP38288 28 2.76E+02 2.00E−65 EP(2)2554 CT10410 6 192 hCG39955 hCT31207hCP49745 5 198 2.00E−16 EP(2)2554 CT10310 15 661 hCG40293 hCT31548hCP50060 18 617 2.00E−90 EP(3)3041 CT5336 14 227 hCG42003 hCT33279hCP51813 29 246 1.00E−39 EP(X)1526 CT10709 7 597 hCG37950 hCT29186hCP47880 6 519 1.00E−168 P1396 = I(2)05206 CT13013 316 655 hCG20435hCT11514 hCP38090 106 404 2.00E−72 P1486 = I(3)00090 CT22943 1818 2491hCG32338 hCT23526 hCP46544 663 1248 2.00E−84 P1505 = I(3)00643 CT23724760 1123 hCG40234 hCT31488 hCP50765 131 439 3.00E−63 P1548 = I(3)01814CT24038 75 167 hCG18539 hCT9598 hCP36359 1 93 1.00E−31 P2093 = I(3)j5C8CT18339 218 438 hCG14845 hCT5866 hCP35211 38 278 6.00E−31 P2093 =I(3)j5C8 CT18415 62 293 bCG17907 hCT8961 hCP33787 353 569 2.00E-18 P2093= I(3)j5C8 CT18415 50 349 hCG41783 hCT33056 hCP51594 7 307 2.00E-23P2104 = (3)j13B3 CT13750 372 733 hCG201263 hCT201265 hCP201588 61 4321.00E−111 P2121 = I(3)j4E1 CT23760 87 283 hCG25031 hCT16153 hCP41935 239437 2.00E−37 P2122 = I(3)rL074 CT23073 5 879 hCG39269 hCT30519 hCP5059221 902 0 P2319 = I(2)06694 CT13966 1 932 hCG21123 hCT12209 hCP38695 18937 0 SEQ ID NO modifier gene name/protein family comments (hCT/hCP)EP(2)2107 TG-interacting factor/TALE/KNOX modifier of Dps and C99 6/7homeobox protein EP(2)2122 n/a modifier of Dps and C99 8/9 EP(2)2151NAP1/aspartyl protease-related modifier of C99 10/11 EP(2)2162 n/amodifier of Dps and C99, lethal over 12/13 C99 EP(2)2173 Drosophila nocAZn finger transcription modifier of Dps and C99, human 14/15, 16/17,factor ortholog ortholog on 10q 52/53 EP(2)2205 anglotensin I convertingenzyme (peptidyl- modifier of Dps and C99, 18/19 dipeptidasemetalloprotease A) 1 (ACE) EP(2)2511 copper chaperone for superoxidedismutase/ modifier of Dps and C99 20/21 superoxide dismutase [CU-ZN]EP(2)2554 glutathione S-transferase theta 1 modifier of Dps and C9922/23 EP(2)2554 intersectin-related modifier of Dps and C99 24/25EP(3)3041 HSA011916 modifier of Dps and C99 26/27 EP(X)1526 proteinkinase Inhibitor P58-related modifier of Dps and C99 28/29 P1396 =I(2)05206 cyclin modifier of Dps and C99 30/31 P1486 = I(3)00090retinoblastoma binding protein-related modifier of Dps and C99 32/33P1505 = I(3)00643 early growth response 2 (Krox-20 modifier of Dps,human ortholog on 34/35 (Drosophila) homolog) 10q P1548 = I(3)01814 n/amodifier of Dps and C99 36/37 P2093 = I(3)j5C8 baculoviral IAPrepeat-containing 4/ modifier of Dps and C99 38/39 apoptosis inhibitorrelated P2093 = I(3)j5C8 ankyrin-related modifier of Dps, human homologon 40/41 10q P2093 = I(3)j5C8 ankyrin-3, ankyrin-G modifier of Dps,human homolog on 42/43 10q P2104 = (3)j13B3 ubiquitin carboxyl-terminalhydrolase modifier of Dps and C99 44/45 P2121 = I(3)j4E1 dualspecificity protein phosphatase modifier of Dps and C99 46/47 P2122 =I(3)rL074 minichromosome maintenance deficient (S. cerevisiae) modifierof Dps and C99 48/49 2 (mitotin)/DNA replication licensing factor MCMP2319 = I(2)06694 alpha-adaptin modifier of C99 50/51

1. A transgenic fly whose genome comprises a DNA sequence encoding apolypeptide comprising the Abeta portion of human APP wherein said DNAsequence encodes Abeta40 (SEQ ID NO:1) or Abeta42 (SEQ ID NO: 2), fusedto a signal sequence, said DNA sequence operably linked to atissue-specific expression control sequence; and expressing said DNAsequence, wherein expression of said DNA sequence results in said flydisplaying an altered phenotype.
 2. A transgenic fly whose genomecomprises a DNA sequence encoding a polypeptide comprising the wild typeC99 portion of human APP (SEQ. ID NO:3) or C99 portion of human APP withthe London Mutation (SEQ ID NO:4), fused to a signal sequence, said DNAsequence operably linked to a tissue-specific expression controlsequence; and expressing said DNA sequence, wherein expression of saidDNA sequence results in said fly displaying an altered phenotype.
 3. Thetransgenic fly of claim 2, wherein said DNA sequence encodes wild typeC99, and wherein said tissue-specific expression control sequencecomprises the UAS control element activated by Gal4 protein produced inthe brain by the 7B-Gal4 transgene.
 4. The transgenic fly of claim 3wherein said expression of said DNA sequence results in said flydisplaying a phenotype characterized as a locomotory defect.
 5. Thetransgenic fly of claim 2, wherein said DNA sequence encodes either wildtype C99 or C99 portion of human APP with the London Mutation, andwherein said tissue-specific expression control sequence is the UAScontrol element activated by Gal4 protein produced by the apterous-Gal4transgene.
 6. The transgenic fly of claim 5 wherein said expression ofsaid DNA sequence results in said fly displaying the “concave wing”phenotype.
 7. A method to identify genetic modifiers of the APP pathway,said method comprising: (a) providing a transgenic fly whose genomecomprises a DNA sequence encoding a polypeptide comprising the Abetaportion of human APP wherein said DNA sequence encodes Abeta40 (SEQ. IDNO:1) or Abeta42 (SEQ ID NO: 2), fused to a signal sequence, said DNAsequence operably linked to a tissue-specific expression controlsequence; and expressing said DNA sequence, wherein expression of saidDNA sequence results in said fly displaying an altered phenotype; (b)crossing said transgenic fly with a fly containing a mutation in a knownor predicted gene; and (c) screening progeny of said crosses for fliesthat carry said DNA sequence and said mutation and display modifiedexpression of the transgenic phenotype as compared to controls.
 8. Themethod of claim 6 wherein said genetic modifier and/or its human homologis a gene that affects the course of Alzheimer's Disease.
 9. The methodof claim 8 wherein said DNA sequence encodes Abeta42, and wherein saidtissue specific expression control sequence comprises the eye-specificpromoter GMR.
 10. The method of claim 8 wherein said expression of saidDNA sequence results in said fly displaying the “rough eye” phenotype.